Gels for encapsulation of biological materials

ABSTRACT

This invention provides novel methods for the formation of biocompatible membranes around biological materials using photopolymerization of water soluble molecules. The membranes can be used as a covering to encapsulate biological materials or biomedical devices, as a “glue” to cause more than one biological substance to adhere together, or as carriers for biologically active species.  
     Several methods for forming these membranes are provided. Each of these methods utilizes a polymerization system containing water-soluble macromers, species which are at once polymers and macromolecules capable of further polymerization. The macromers are polymerized using a photoinitiator (such as a dye), optionally a cocatalyst, optionally an accelerator, and radiation in the form of visible or long wavelength UV light. The reaction occurs either by suspension polymerization or by interfacial polymerization. The polymer membrane can be formed directly on the surface of the biological material, or it can be formed on material which is already encapsulated.

BACKGROUND

[0001] Microencapsulation technology holds promise in many areas ofmedicine. For example, some important applications are treatment ofdiabetes (Goosen, et al., 1985), production of biologically importantchemicals (Omata, et al., 1979), evaluation of anti-humanimmunodeficiency virus drugs (McMahon, et al., 1990), encapsulation ofhemoglobin for red blood cell substitutes, and controlled release ofdrugs. During encapsulation using prior methods, cells are often exposedto processing conditions which are potentially cytotoxic. Theseconditions include heat, organic solvents and non-physiological pH whichcan kill or functionally impair cells. Proteins are often exposed toconditions which are potentially denaturing and can result in loss ofbiological activity.

[0002] Further, even if cells survive processing conditions, thestringent requirements of encapsulating polymers for biocompatibility,chemical stability, immunoprotection and resistance to cellularovergrowth, restrict the applicability of prior art methods. Forexample, the encapsulating method based on ionic crosslinking ofalginate (a polyanion) with polylysine or polyornithine (polycation)(Goosen, et al., 1987) offers relatively mild encapsulating conditions,but the long-term mechanical and chemical stability of such tonicallycrosslinked polymers remains doubtful. Moreover, these polymers whenimplanted in vivo, are susceptible to cellular overgrowth (McMahon, etal., 1990) which restricts the permeability of the microcapsule tonutrients, metabolites, and transport proteins from the surroundings.This has been seen to possibly lead to starvation and death ofencapsulated islets of Langerhans cells (O'Shea and Sun, 1986).

[0003] Thus, there is a need for a relatively mild cell encapsulationmethod which offers control over properties of the encapsulatingpolymer. The membranes must be non-toxically produced in the presence ofcells, with the qualities of being permselective, chemically stable, andvery highly biocompatible. A similar need exists for the encapsulationof biological materials other than cells and tissues.

[0004] Biocompatibility

[0005] Synthetic or natural materials intended to come in contact withbiological fluids or tissues are broadly classified as biomaterials.These biomaterials are considered biocompatible if they produce aminimal or no adverse response in the body. For many uses ofbiomaterials, it is desirable that the interaction between thephysiological environment and the material be minimized. For these uses,the material is considered “biocompatible” if there is minimal cellulargrowth on its surface subsequent to implantation, minimal inflammatoryreaction, and no evidence of anaphylaxis during use. Thus, the materialshould elicit neither a specific humoral or cellular immune response nora nonspecific foreign body response.

[0006] Materials which are successful in preventing all of the aboveresponses are relatively rare; biocompatibility is more a matter ofdegree rather than an absolute state. The first event occurring at theinterface of any implant with surrounding biological fluids is proteinadsorption (Andrade, et al., 1986). In the case of materials of naturalorigin, it is conceivable that specific antibodies for that materialexist in the repertoire of the immune defense mechanism of the host. Inthis case a strong immune response can result. Most synthetic materials,however, do not elicit such a reaction. They can either activate thecomplement cascade or adsorb serum proteins which mediate cell adhesion,called cell adhesion molecules (CAMs) (Buck, et al., 1987). The CAMfamily includes proteins such as fibronectin, vitronectin, laminin, vonWillebrand factor, and thrombospondin.

[0007] Proteins can adsorb on almost any type of material. They havepositively and/or negatively charged regions, as well as hydrophilic andhydrophobic regions. They can thus interact with implanted materialthrough any of these various regions, resulting in cellularproliferation at the implant surface. Complement fragments such as C3bcan be immobilized on the implant surface and act as chemoattractants.They in turn can activate inflammatory cells such as macrophages andneutrophils and cause their adherence and activation on the implant.These cells attempt to degrade and digest the foreign material.

[0008] In the event that the implant is nondegradable and is too largeto be ingested by large single activated macrophages, the inflammatorycells may undergo frustrated phagocytosis. Several such cells cancombine to form foreign body giant cells. In this process, these cellsrelease peroxides, hydrolytic enzymes, and chemoattractant andanaphylactic agents such as interleukins, which increase the severity ofthe reaction. They also induce the proliferation of fibroblasts onforeign surfaces.

[0009] Fibroblasts secrete a collagenous matrix which ultimately resultsin encasement of the entire implant in a fibrous envelope. Cell adhesioncan also be mediated on a charged surface by the cell surfaceproteoglycans such as heparin sulfate and chondroitin sulfate (vanWachem, et al., 1987). In such a process, intermediary CAMs are notrequired and the cell surface can interact directly with the surface ofthe implant.

[0010] Enhancing Biocompatibility

[0011] Past approaches to enhancing biocompatibility of materialsstarted with attempts at minimization of interfacial energy between thematerial and its aqueous surroundings. Similar interfacial tensions ofthe solid and liquid were expected to minimize the driving force forprotein adsorption and this was expected to lead to reduced celladhesion and thrombogenicity of the surface. For example, Amudeshwari etal. used collagen gels cross-linked in the presence of HEMA and MMX(Amudeshwari, et al., 1986). Desai and Hubbell showed a poly(HEMA)-MMAcopolymer to be somewhat non-thrombogenic (Desai, N. P. and Hubbell,1989).

[0012] Protein adsorption and desorption, however, is a dynamicphenomenon, as seen in the Vroman effect. This effect is the gradualdisplacement of one serum protein by another, through a well-definedseries, until only virtually irreversibly adsorbed proteins are presenton the surface. Affinity of protein in a partially dehydrated state forthe polymer surface has been proposed as a determining factor forprotein adsorption onto a surface (Baier, 1990). Enhancement of surfacehydrophilicity has resulted in mixed success; increased hydrophilicityor hydrophobicity does not have a clear relation with biocompatibility(Coleman, et al., 1982; Hattori, et al., 1985). In some cases, surfaceswith intermediate hydrophilicities demonstrate proportionately lessprotein adsorption. The minimization of protein adsorption may dependboth upon hydrophilicity and the absence of change, as described furtherbelow, perhaps in addition to other factors.

[0013] Use of Gels in Biomaterials

[0014] Gels made of polymers which swell in water such as poly (HEMA),water-insoluble polyacrylates, and agarose, have been shown to becapable of encapsulating islet cells and other animal tissue (Iwata, etal., 1989; Lamberti, et al., 1984). However, these gels have undesirablemechanical properties. Agarose forms a weak gel, and the polyacrylatesmust be precipitated from organic solvents, thus increasing thepotential for cytotoxicity. Dupuy et al. (1988) have reported themicroencapsulation of islets by polymerization of acrylamide to formpolyacrylamide gels. However, the polymerization process, if allowed toproceed rapidly to completion, generates local heat and requires thepresence of toxic cross-linkers. This usually results in mechanicallyweak gels whose immunoprotective ability has not been established.Moreover, the presence of a low molecular weight monomer is requiredwhich itself is cytotoxic.

[0015] Microcapsules formed by the coacervation of alginate and poly(L-lysine) have been shown to be immunoprotective e.g., O'Shea et al.,1986. However, implantation for periods up to a week has resulted insevere fibrous overgrowth on these microcapsules (McMahon, et al. 1990;O'Shea, et al., 1986).

[0016] Use of PEO in Biomaterials

[0017] The use of poly(ethylene oxide) (PEO) to increasebiocompatibility is well documented in the literature. The presence ofgrafted PEO on the surface of bovine serum albumin has been shown byAbuchowski et al. (1977) to reduce immunogenicity in a rabbit and toincrease circulation times of exogenous proteins in animals. Thebiocompatibility of algin-poly(L-lysine) microcapsules has beensignificantly enhanced by incorporating a graft copolymer of PLL and PEOon the microcapsule surface (Sawhney, et al. in press).

[0018] The grafting of methoxy PEO onto polyacrylonitrile surfaces wasseen by Miyama et al. (1988) to render the polyacrylonitrile surfacerelatively non-thrombogenic. Nagoaka et al. synthesized a graftcopolymer of methacrylates with PEO and found the resulting polymer tobe highly non-thrombogenic. Desai and Hubbell have immobilized PEO onpoly(ethylene terepthalate) surfaces by forming a physicalinterpenetrating network (Desai et al., 1992); they have shown thesesurface to be highly resistant to thrombosis (Desai et al, 1991) and toboth mammalian and bacterial cell growth (Desai, et al., submitted).

[0019] PEO is a unique polymer in terms of structure. The PEO chain ishighly water soluble and highly flexible. Polymethylene glycol, on theother hand, undergoes rapid hydrolysis, while polypropylene oxide isinsoluble in water. PEO chains have an extremely high motility in waterand are completely non-ionic in structure. The synthesis andcharacterization of PEO derivatives which can be used for attachment ofPEO to various surfaces, proteins, drugs etc. has been reviewed (Harris,1985). Other polymers are also water soluble and non-ionic, such aspoly(N-vinyl pyrrolidinone) and poly(ethyl oxazoline). These have beenused to reduce interaction of cells with tissues. N. P. Desai et al.(1991). Water soluble ionic polymers, such as hyaluronic acid, have alsobeen used to reduce cell adhesion to surfaces and can similarly be used.

[0020] Immobilization of PEO on a charged surface, such as a coacervatedmembrane of alginate-PLL, results in shielding of surface charges by thenon-ionic PEO (Sawhney et al., in press). The highly motile PEO chainsweeps out a free volume in its microenvironment. The free volumeexclusion effect makes the approach of a macromolecule (viz., a protein)close to a surface which has grafted PEO chains sterically unfavorable(Miyama, et al., 1988; Nagoaka, et al.; Desai, et al.: Sun, et al.,1987). Thus protein adsorption is minimized and cell adhesion isreduced, resulting in surfaces showing increased biocompatibility.

[0021] Immobilization of PEO on a surface has been largely carried outby the synthesis of graft copolymers having PEO side chains (Sawhney, etal.; Miyama, et al., 1988; Nagoaka, et al.) This process involves thecustom synthesis of monomers and polymers for each application. The useof graft copolymers, however, still does not guarantee that the surface“seen” by a macromolecule consists entirely of PEO.

[0022] Electron beam cross-linking has been used to synthesize PEOhydrogels, and these biomaterials have been reported to benon-thrombogenic (Sun, et al., 1987; Dennison, H. A. 1986). However, useof an electron beam precludes the presence of any living tissue due tothe sterilizing effect of this radiation. Also, the networks producedare difficult to characterize due to he non-specific cross-linkinginduced by the electron beam.

[0023] Photopolymerizable, PEG liacrylates have been used to entrapyeast cells for fermentation and chemical conversion (Kimura et al.1981: Omata et al., 1981; Okada et al. 1987). However, yeast cells arewidely known to be much hardier, resistant to adverse environments andelevated temperatures, and more difficult to kill when compared tomammalian cells and human tissues. For example, yeast may be grownanaerobically, whereas mammalian cells may not; yeast are more resistantto organic solvents (e.g., ethanol to 12%) than are mammalian cells(e.g., ethanol to <1%); and yeast possess a polysaccharide cell wall,whereas mammalian cells, proteins, polysaccharides, and drugs do not.None of these references, however, discuss the exposure of sensitiveeukaryotic tissue, organisms, or sensitive molecules to the chemicalconditions used during polymerization because their polymerizationconditions are incompatible with sensitive materials. For example, thereare no reports of the encapsulation of mammalian cells using prior artphotosensitive prepolymers without a marked loss of cellular function.

[0024] Other earlier encapsulations of cells within photopolymerizablematerials have focused on microbial cells (Kimura et al., 1981; Omata etal., 1981; Okada et al., 1987; Tanaka et al. 1977; Omata et al1979a .;Omata et al., 1979a; Omata et al., 1979b; Chun et al. 1981; Fukui etal., 1976; Fukui et al., 1984). Each of these reports, however,describes the use of near ultraviolet light (wavelength <320 nm), whichis injurious to more sensitive cells such as mammalian cells or highereukaryotic cells. In the original presentation of the technique (Fukuiet al., 1976), the authors state in the final sentence that thetechnique would be appropriate for microbial cells, but provide noindication of usefulness for more sensitive cells. In a more recent andcomplete review of the technique (Fukui et al., 1984), the authors, insection 6 entitled “Entrapped Living Cells” provide no teachingregarding cells other than microbial cells, and in section 7 entitled“Future Prospects” they also provide no such teaching.

[0025] Moreover, the prior use of such materials for the entrapment ofbiological materials is entirely focused on industrial technology,rather than biomedical technology. For example, no attention is paid tobiocompatibility, including formulation of the gel to avoid the problemsdescribed above. This is an important issue, since bioincompatibility inbiomedical applications leads to xenograft failure in therapeuticallytransplanted cells for the evaluation of drug efficacy (O'Shea et al.,1986) and to xenograft failure in diagnostically transplanted cells(McMahon et al., 1990). Similarly, bioincompatibility would lead to thefailure of encapsulated enzymes (for example, therapeutic enzymesencapsulated and circulating or implanted in a blood-rich tissue). Suchencapsulated and entrapped enzymes could leave the circulation byinteraction with the reticuloendothelial system (Hunt et al., 1985) orcould become overgrown with tissues in a foreign body reaction.

[0026] Other ways of producing PEO hydrogels include use of PEO chainsend capped with n-alkane chains, which associate in aqueous media toform stable gels (Knowles, et al., 1990). No biological properties ofthese materials have been reported, however. Thus, the prior artcontains no description of methods to form biocompatible PEO networks onthree-dimensional living tissue surfaces without damaging encapsulatedtissue.

[0027] Among the techniques for encapsulating mammalian tissue withpolymers other than PEO is a method of photopolymerizing the monomer2-hydroxyethyl methacrylate (“HEMA”) and the crosslinking agent ethyleneglycol dimethacrylate (“EGDA”) in a cylindrical mold containing thebiological material (Ronel, et al., 1981). The product of this reaction,a cylindrical gel with cells embedded throughout, is frozen and thenfinely ground into small particles. This technique, however, suffersfrom a number of disadvantages. First, because the cylindrical gel isbroken along random planes, shearing will often occur through pockets ofcells, leaving some cells exposed to the host immune system. Second,HEMA and EGDA are small cytotoxic molecules capable of penetrating thecellular membrane. Third, the resulting polymer membrane has uneven poresizes which vary to an upper limit of 20 microns, thereby allowingtransit of immune response molecules. These drawbacks are reflected indata which show that tissue remains viable for only 2-3 days after thisencapsulation process.

SUMMARY OF THE INVENTION

[0028] This invention provides novel methods for the formation ofbiocompatible membranes around biological materials usingphotopolymerization of water soluble molecules. The membranes can beused as a covering to encapsulate biological materials or biomedicaldevices, as a “glue” to cause more than one biological substance toadhere together, or as carriers for biologically active species.

[0029] Several methods for forming these membranes are provided. Each ofthese methods utilizes a polymerization system containing water-solublemacromers, species which are at once polymers and macromolecules capableof further polymerization. The macromers are polymerized using aphotoinitiator (such as a dye), optionally a cocatalyst, optionally anaccelerator, and radiation in the form of visible or long wavelength UVlight. The reaction occurs either by suspension polymerization or byinterfacial polymerization. The polymer membrane can be formed directlyon the surface of the biological material, or it can be formed onmaterial which is already encapsulated.

[0030] Ultrathin membranes can be formed by the methods describedherein. These ultrathin membranes allow for optimal diffusion ofnutrient and bioregulator molecules across the membrane, and greatflexibility in the shape of the membrane. Such thin membranes produceencapsulated material with optimal economy of volume. Biologicalmaterial thus coated can be packed into a relatively small space withoutinterference from bulky membranes.

[0031] The thickness and pore size of membranes formed can be varied.This variability allows for “perm-selectivity”—membranes can be adjustedto the desired degree of porosity, allowing only preferred molecules topermeate the membrane, while acting as a barrier against largerundesired molecules. Thus, the membranes are immunoprotective in thatthey prevent the transfer of antibodies or cells of the immune system.

[0032] When the encapsulated biological material is cellular in nature,the absence of small monomers in the polymerization solution preventsthe diffusion of toxic molecules into the cell. In this manner thepresent invention provides a polymerization system which is morebiocompatible than any available in the prior art.

[0033] Additionally, the polymerization method utilizes short bursts ofvisible or long wavelength UV light which is nontoxic to biologicalmaterial. Bioincompatible polymerization initiators employed in theprior art are also eliminated.

[0034] According to the present invention, membrane formation occursunder physiological conditions. Thus, no damage is done to the enclosedbiological material due to harsh pH, temperature, or ionic conditions.

[0035] Because the membrane adheres to the biological material, themembrane can be used as an adhesive to fasten more than one biologicalsubstance together. The macromers are polymerized in the presence ofthese substances which are in close proximity. The membrane forms in theinterstices, effectively gluing the substances together.

[0036] Additionally, utilizing the tendency of the membrane to adhere tobiological material, a membrane can be formed around or on abiologically active substance to act as a carrier for that substance.

DETAILED DESCRIPTION

[0037] By a variety of methods, this invention provides a means forcreating biocompatible membranes of varying thickness on the surface ofa variety of biological materials. The polymerization occurs by afree-radical reaction, causing a “macromer” with at least twoethylenically unsaturated moieties to form a crosslinked polymer. Thecomponents of this reaction are:

[0038] (1) a photoinitiator, preferably eosin dye;

[0039] (2) a “macromer,” preferably polyethylene glycol (PEG)diacrylate, m.w. 18.5 kD. This component is at once a polymer and amacromolecule capable of further polymerization;

[0040] (3) optionally a cocatalyst, preferably triethanolamine; and

[0041] (4) optionally, an accelerator.

[0042] These components are mixed in varying combinations, and themixture is exposed to longwave UV or visible light (“radiation”),preferably of wavelength 350-700 nm, most preferred at 365-514 nm, toinitiate polymerization. A network is formed as the macromers polymerizein a variety of directions.

[0043] Four methods are used to effect polymerization to formbiocompatible membranes. These are referred to below an the “bulksuspension polymerization” method, the “microcapsule suspensionpolymerization” method, the “microcapsule interfacial polymerization”method, and the “direct interfacial polymerization” method. They utilizeeither suspension or interfacial polymerization techniques on eithercoated or uncoated biological material.

[0044] Bulk Suspension Polymerization Method

[0045] In this embodiment of the invention the core biological materialis mixed in an aqueous macromer solution (composed of the macromer,cocatalyst and optionally an accelerator) with the photoinitiator. Smallglobular geometric structures such as spheres, ovoids, or oblongs areformed, preferably either by coextrusion of the aqueous solution withair or with a non-miscible substance such as oil, preferably mineraloil, or by agitation of the aqueous phase in contact with a non-misciblephase such as an oil phase to form small droplets. The macromer in theglobules is then polymerized when exposed to radiation. Because themacromer and initiator are confined to the globules, the structureresulting from polymerization is a capsule in which the biologicalmaterial is enclosed. This is a “suspension polymerization” whereby theentire aqueous portion of the globule polymerizes to form a thickmembrane around the cellular material.

[0046] Microcapsule Suspension Polymerization Method

[0047] This embodiment of the invention employs microencapsulatedmaterial as a core about which the macromer is polymerized in asuspension polymerization reaction. The biological material is firstencapsulated, such as in an alginate microcapsules. The microcapsule isthen mixed as in the first embodiment with the macromer solution and thephotoinitiator, and then polymerized by radiation.

[0048] This method takes advantage of the extreme hydrophilicity of PEGmacromer, and is especially suited for use with hydrogel microcapsulessuch as alginate-poly(L-lysine). The microsphere is swollen in water.When a macromer solution (with the initiating system) is forced to phaseseparate in a hydrophobic medium, such as mineral oil, the PEG macromersolution prefers to stay on the hydrophilic surface of the alginatemicrocapsule. When this suspension is irradiated, the PEG macromerundergoes polymerization and gelation, forming a thin layer ofpolymeric, water insoluble gel around the microsphere. Agarose beadshave been used in an analogous way by Gin et al. (1987) as scaffolds tocarry out polymerization of acrylamide. However, that method is limitedby potential toxicity associated with the use of a low molecular weightmonomer, as opposed to the macromeric precursors of the presentinvention.

[0049] This technique preferably involves coextrusion of themicrocapsule in a solution of macromer and photoinitiator, the solutionbeing in contact with air or a liquid which is non-miscible with water,to form droplets which fall to a container such as a petri dishcontaining a solution such as mineral oil in which the droplets are notmiscible. The non-miscible liquid is chosen for its ability to maintaindroplet formation. Additionally, if the membrane-encapsulated materialis to be injected or implanted in an animal, any residue should benon-toxic and non-immunogenic. Mineral oil is a preferred non-miscibleliquid.

[0050] On the petri dish the droplets are exposed to radiation whichcauses polymerization. This coextrusion technique results in acrosslinked polymer coat of greater than 50 microns thickness.Alternatively, the microcapsules may be suspended in a solution ofmacromer and photoinitiator which is agitated in contact with anon-miscible phase such as an oil phase. The emulsion which results isirradiated to form a polymer coat, again of greater than 50 micronsthickness.

[0051] Microcapsule Interfacial Polymerization Method

[0052] In this embodiment, the biological material is alsomicroencapsulated as in the previous method. However, rather thansuspension polymerization, interfacial polymerization is utilized toform the membrane. This involves coating the microcapsule withphotoinitiator, suspending the microcapsule in the macromer solution,and immediately irradiating. By this technique a thin polymer coat, ofless than 50 microns thickness, is formed about the microcapsule,because the photoinitiator is present only at the microcapsule surfaceand is given insufficient time to diffuse far into the macromersolution. As a result, the initiator is present in only a thin shell ofthe aqueous solution, causing a thin layer to be polymerized.

[0053] When the microcapsules are in contact with dye solution, the dyepenetrates into the inner core of the microcapsule as well as adsorbingto the surface. When such a microcapsule is put into a solutioncontaining a macromer and, optionally, a cocatalyst such astriethanolamine, and exposed to laser light, initially all the essentialcomponents of the reaction are present only at and just inside theinterface of microcapsule and macromer solution. Hence, thepolymerization and gelation (if multifunctional macromer is used)initially takes place only at the interface, just beneath it, and justbeyond it. If left for longer periods of time, the dye starts diffusingfrom the inner core of the microsphere into the solution; similarly,macromers start diffusing inside the core.

[0054] Polymerization and subsequent gelation are very rapid (typicalgelation times are 100 ms) (Fouassier, et al., 1985; Chesneau, et al.,1985). Because diffusion is a much slower process than polymerization,not the entire macromer solution is polymerized or gelled. Essentiallythe reaction is restricted to the near surface only. The dye, being asmaller molecule and being weakly bound to the capsule materials, keepsdiffusing out of the microsphere. If this diffusion occurs under laserirradiation, then dye at the interface is used continuously to form athicker gel layer. The thickness of the coating can thus be directed bycontrolling the reaction conditions.

[0055] A schematic representation of this process is shown in FIG. 2A.The amount, thickness or size and rigidity of the gel formed will dependon the size and intensity of the beam, time of exposure, initiator,macromer molecular weight, and macromer concentration (see below).Alginate/PLL microspheres containing islets coated by this technique areshown in FIG. 2B.

[0056] Direct Interfacial Polymerization Method

[0057] The fourth embodiment of this invention utilizes interfacialpolymerization to form a membrane directly on the surface of thebiological material. This results in the smallest capsules and thusachieves optimal economy of volume. Tissue is directly coated withphotoinitiator, emersed in the macromer solution, and immediatelyirradiated. This technique results in a thin polymer coat surroundingthe tissue since there is no space taken up by a microcapsule, and thephotoinitiator is again present only in a thin shell of the macromersolution.

[0058] Use as an Adhesive

[0059] It is usually difficult to get good adhesion between polymers ofgreatly different physicochemical properties. The concept of a surfacephysical interpenetrating network was presented by Desai and Hubbel (N.P. Desai et al. (1992)). This approach to incorporating into the surfaceof one polymer a complete coating of a polymer of considerably differentproperties involved swelling the surface of the polymer to be modified(base polymer) in a mutual solvent, or a swelling solvent, for the basepolymer and for the polymer to be incorporated (penetrant polymer). Thepenetrant polymer diffused into the surface of the base polymer. Thisinterface was stabilized by rapidly precipitating or deswelling thesurface by placing the base polymer in a nonsolvent bath. This resultedin entanglement of the penetrant polymer within the matrix of the basepolymer at its surface in a structure that was called a surface physicalinterpenetrating network.

[0060] This approach can be improved upon by photopolymerizing thepenetrant polymer upon the surface of the base polymer in the swollenstate. This results in much enhanced stability over that of the previousapproach and in the enhancement of biological responses to thesematerials. The penetrant may be chemically modified to be a prepolymer(macromer), i.e. capable of being polymerized itself. Thispolymerization can be initiated thermally or by exposure to visible,ultraviolet, infrared, gamma ray, or electron beam irradiation, or toplasma conditions. In the case of the relatively nonspecific gamma rayor electron beam radiation reaction, chemical incorporation ofparticularly reactive sites may not be necessary.

[0061] Polyethylene glycol (PEG) is a particularly useful penetrantpolymer for biomedical applications where the lack of cell adhesion isdesired. The previous work had demonstrated an optimal performance at amolecular weight of 18,500 D without chemical crosslinking. PEGprepolymers can be readily formed by acrylation of the hydroxyl groupsat its termini or elsewhere within the chain. These prepolymers can bereadily polymerized by the above described radiation methods.Photoinititated polymerization of these prepolymers is particularlyconvenient and rapid. There are a variety of visible light initiated andultraviolet light initiated reactions that are initiated by lightabsorption by specific photochemically reactive dyes, describedelsewhere herein. This same approach can be used for biomedical purposeswith other water-soluble polymers, such as poly(N-vinyl pyrrolidinone),poly(N-isopropyl acrylamide), poly(ethyl oxazoline) and many others.

[0062] Additionally, it is usually difficult to obtain adhesives for wetsurfaces and tissues. Water soluble prepolymers, for example PEGdiacrylates, can be used for this purpose. When a water soluble polymeris placed in aqueous solution upon a tissue, the polymer diffuses intothe surface of the tissue, within the protein and polysaccharide matrixupon the tissue but not within the cells themselves. When the watersoluble polymer is a prepolymer and a visible, ultraviolet or infraredphotoinitiator is included, the polymer penetrant may be exposed to theappropriate light to gel the polymer. In this way, the polymer iscrosslinked within and around the matrix of the tissue in what is calledan interpenetrating network. If the prepolymer is placed in contact withtwo tissues and the prepolymer is illuminated, then these two tissuesare adhered together by the intermediate polymer gel.

[0063] Biological Materials

[0064] Due to the biocompatibility of the materials and techniquesinvolved, a wide variety of materials can be used in conjunction withthe present invention. For encapsulation, the techniques can be usedwith mammalian tissue and/or cells, as well as sub-cellular organellesand other isolated sub-cellular components. The membranes can be craftedto meet the permselectively needs of the biological material enclosed.Cells which are to be used to produce desired products such as proteinsare optimally encapsulated by this invention.

[0065] Examples of cells which can be encapsulated are primary culturesas well as established cell lines, including transformed cells. Theseinclude but are not limited to pancreatic islet cells, human foreskinfibroblasts, Chinese hamster ovary cells, beta cell insulomas,lymphoblastic leukemia cells, mouse 3T3 fibroblasts, dopamine secretingventral mesencephalon cells, neuroblastoid cells, adrenal medulla cells,and T-cells. As can be seen from this partial list, cells of all types,including dermal, neural, blood, organ, muscle, glandular, reproductive,and immune system cells can be encapsulated successfully by this method.Additionally, proteins (such as hemoglobin), polysaccharides,oligonucleotides, enzymes (such as adenosine deaminase), enzyme systems,bacteria, microbes, vitamins, cofactors, blood clotting factors, drugs(such as TPA, streptokinase or heparin), antigens for immunization,hormones, and retroviruses for gene therapy can be encapsulated by thesetechniques.

[0066] The biological material can be first enclosed in a structure suchas a polysaccharide gel. (Lim, U.S. Pat. No. 4,352,883; Lim, U.S. Pat.No. 4,391,909; Lim, U.S. Pat. No. 4,409,331; Tsang, et al., U.S. Pat.No. 4,663,286; Goosen et al., U.S. Pat. No. 4,673,556; Goosen et al.,U.S. Pat. No. 4,689,293; Goosen et al., U.S. Pat. No. 4,806,355; Rha etal., U.S.Pat. No. 4,744,933; Rha et al., U.S. Pat. No. 4,749,620,incorporated herein by reference.) Such gels can provide additionalstructural protection to the material, as well as a secondary level ofperm-selectivity.

[0067] Macromers

[0068] Polymerization via this invention utilizes macromers rather thanmonomers as the building blocks. The macromers are small polymers whichare susceptible to polymerization into the larger polymer membranes ofthis invention. Polymerization is enabled because the macromers containcarbon-carbon double bond moieties, such as acrylate, methacrylate,ethacrylate, 2-phenyl acrylate, 2-chloro acrylate, 2-bromo acrylate,itaconate, acrylamide, methacrylamide, and styrene groups. The macromersare water soluble compounds and are non-toxic to biological materialbefore and after polymerization.

[0069] Examples of macromers are ethylenically unsaturated derivativesof poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG), poly(vinylalcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline)(PEOX), poly(amino acids), polysaccharides such as alginate, hyaluronicacid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparinsulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum,water soluble cellulose derivatives and carrageenan, and proteins suchas gelatin, collagen and albumin. An example of a macromer is

[0070] where

[0071] F₁=CONH, COO or NHCOO

[0072] X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOH

[0073] F₂=COO, CONH, O or C₆H₄,

[0074] R=CH₂ or-alkyl-,

[0075] n≧5, and

[0076] m≧3.

[0077] These macromers can vary in molecular weight from 0.2-100 kD,depending on the use. The degree of polymerization, and the size of thestarting macromers, directly affect the porosity of the resultingmembrane. Thus, the size of the macromers are selected according to thepermeability needs of the membrane. For purposes of encapsulating cellsand tissue in a manner which prevents the passage of antibodies acrossthe membrane but allows passage of nutrients essential for cellularmetabolism, the preferred starting macromer size is in the range of 10kD to 18.5 kD, with the most preferred being around 18.5 kD. Smallermacromers result in polymer membranes of a higher density with smallerpores.

[0078] Photoinitiating Dyes

[0079] The photoinitiating dyes capture light energy and initiatepolymerization of the macromers. Any dye can be used which absorbs lighthaving frequency between 320 nm and 900 nm, can form free radicals, isat least partially water soluble, and is non-toxic to the biologicalmaterial at the concentration used for polymerization. Examples ofsuitable dyes are ethyl eosin, eosin Y, fluorescein,2,2-dimethoxy,2-phenylacetophenone, 2-methoxy,2-phenylacetophenone,camphorquinone, rose bengal, methylene blue, erythrosin, phloxime,thionine, riboflavin and methylene green. The preferred initiator dye isethyl eosin due to its spectral properties in aqueous solution.

[0080] Cocatalyst

[0081] The cocatalyst is a nitrogen based compound capable ofstimulating the free radical reaction. Primary, secondary, tertiary orquaternary amines are suitable cocatalysts, as are any nitrogen atomcontaining electron-rich molecules. Cocatalysts include, but are notlimited to, triethanolamine, triethylamine, ethanolamine, N-methyldiethanolamine, N,N-dimethyl benzylamine, dibenzyl amine, N-benzylethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine,potassium persulfate, tetramethyl ethylenediamine, lysine, ornithine,histidine and arginine.

[0082] Radiation Wavelength

[0083] The radiation used to initiate the polymerization is eitherlongwave UV or visible light, with a wavelength in the range of 320-900nm. Preferably, light in the range of 350-700 nm, and even morepreferred in the range of 365-514 nm, is used. This light can beprovided by any appropriate source able to generate the desiredradiation, such as a mercury lamp, longwave UV lamp, He—Ne laser, or anargon ion laser.

[0084] Thickness and Conformation of Polymer Layer

[0085] Membrane thickness affects a variety of parameters, includingperm-selectivity, rigidity, and size of the membrane. In the interfacialpolymerization method, the duration of the radiation can be varied toadjust the thickness of the polymer membrane formed. This correlationbetween membrane thickness and duration of irradiation occurs becausethe photoinitiator diffuses at a steady rate, with diffusion being acontinuously occurring process. Thus, the longer the duration ofirradiation, the more photoinitiator will initiate polymerization in themacromer mix, the more macromer will polymerize, and a thicker coat willbe formed. Additional factors which affect membrane thickness are thenumber of reactive groups per macromer, the concentration ofaccelerators in the macromer solution. This technique allows thecreation of very thin membranes because the photoinitiator is firstpresent in a very thin layer at the surface of the biological material,and polymerization only occurs where the photoinitiator is present.

[0086] The suspension polymerization method forms a somewhat thickermembrane than the interfacial polymerization method. This is becausepolymerization occurs in the suspension method throughout the macromermix. The thickness of membranes formed by the suspension method isdetermined in part by the viscosity of the macromer solution, theconcentration of the macromer in that solution, the fluid mechanicalenvironment of the suspension and surface active agents in thesuspension. These membranes vary in thickness from 50-300 microns. Theshape of the structure formed by suspension polymerization can becontrolled by shaping the reaction mix prior to polymerization. Spherescan be formed by emulsion with a non-miscible liquid such as oil,coextrusion with such a liquid, or coextrusion with air. Cylinders maybe formed by casting or extrusion, and slabs and discoidal shapes can beformed by casting. Additionally, the shape may be formed in relationshipto an internal supporting structure such as a screening network ofstable polymers (e.g. an alginate gel or a woven polymer fiber) ornontoxic metals.

[0087] The overall amount, thickness, and rigidity of the membraneformed depends on the interaction of several parameters, including thesize and intensity of the radiation beam, duration of exposure of thesolution to the radiation, reactivity of the initiator selected,macromer molecular weight, and macromer concentration.

[0088] The invention can be used for a variety of purposes, some ofwhich are enumerated below, along with benefits which accrue from theuse of the invention:

[0089] a. Microencapsulating cells: more biocompatible, stronger, morestable, better control of permselectivity, less toxic conditions

[0090] b. Macroencapsulating cells: more biocompatible, stronger, morestable, better control of permselectivity, less toxic conditions, easierto incorporate internal or external supporting structure

[0091] c. Microencapsulating or macroencapsulating other tissues, withthe same benefits

[0092] d. Microencapsulating or macroencapsulating pharmaceuticals: morebiocompatible, less damaging to the pharmaceutical

[0093] e. Coating devices: ease of application, more biocompatible

[0094] f. Coating microcapsules: more biocompatible, strengthens them,ease of coating

[0095] g. Coating macrocapsules, microcapsules, microspheres andmacrospheres: more biocompatible, ease of coating

[0096] h. Coating tissues to alter adhesion of other tissues: ease ofcoating, less toxicity to the tissues, conformal coating versusnonconformal

[0097] i. Adhesive between two tissues: ease of adhesion, rapidity offorming adhesive bond, less toxicity to tissues

[0098] The invention described herein is further exemplified in thefollowing Examples. While these Examples provide a variety ofcombinations useful in performing the methods of the invention, they areillustrative only and are not to be viewed as limiting in any manner thescope of the invention.

[0099] Example 1—Synthesis of PEG 6 kD Diacrylate

[0100] Example 2—Synthesis of PEG 18.4 kD Tetraacrylate

[0101] Example 3—Coating of Islet-containing Alginate-PLL Microspheresby Surface Dye Adsorption

[0102] Example 4—Coating Islet-containing Alginate-PLL Microspheres bythe Oil Suspension Method

[0103] Example 5—Encapsulation of Islets of Langerhans

[0104] Example 6—Microencapsulation of Animal Cells

[0105] Example 7—Coating of Animal Cell-Containing Alginate-PLLMicrospheres and Individual Cells by Surface Dye Adsorption

[0106] Example 8—Coating Animal Cell Containing Alginate-PLLMicrospheres by the Oil Suspension Method

[0107] Example 9—Coating of Individual Islets of Langerhans by SurfaceDye Adsorption

[0108] Example 10—Biocompatibility of PEO on Microspheres

[0109] Example 11—Permeability of PEO Gels

[0110] Example 12—Treatment of Silicone Rubber

[0111] Example 13—Treatment of Polyurethane

[0112] Example 14—Treatment of Ultrafiltration Membranes

[0113] Example 15—Treatment of Textured Materials and Hydrogels

[0114] Example 16—Treatment of Dense Materials

[0115] Example 17—Rate of Polymerization

[0116] Example 18—PEO Gel Interactions

[0117] Example 19—Characterization and Mechanical Analysis of PEO Gels

[0118] Example 20—Water Content of PEO Gels

[0119] Example 21—Mechanical Stability of PEO Gels after Implantation

[0120] Example 22—Monitoring of Calcification of PEO Gels

[0121] Example 23—Encapsulation of Neurotransmitter-Releasing Cells

[0122] Example 24—Encapsulation of Hemoglobin for Synthetic Erythrocytes

[0123] Example 25—Entrapment of Enzymes for Correction of MetabolicDisorders and Chemotherapy

[0124] Example 26—Cellular Microencapsulation for Evaluation ofAnti-Human Immunodeficiency Virus Drugs In Vivo

[0125] Example 27—Use of PEG Gels as Adhesive to Rejoin Severed Nerve

[0126] Example 28—Surgical Adhesive

[0127] Example 29—Modification of PVA Polymer

[0128] Example 30—Use of Alternative Photopolymerizable Moieties

[0129] Example 31—Use of Alternative Photoinitiator/PhotosensitizerSystems

[0130] Example 32—Formation of Alginate-PLL-Alginate Microcapsules withPhotopolymerizable Polycations

EXAMPLE 1

[0131] Synthesis of PEG 6 kD Diacrylate

[0132] PEG acrylates of molecular weights 400 Da and 1,000 Da warecommercially available from Sartomer and Dajac Inc., respectively. PEG 6kD (20 g) was dissolved in 200 mL dichloromethane in a 250 mL roundbottom flask. The flask was cooled to 0° C. and 1.44 mL of triethylamine and 1.3 mL of acryloyl chloride were added with constant stirringunder a dry nitrogen atmosphere. The reaction mixture was then broughtto room temperature and stirred for 12 hr under a nitrogen atmosphere.It was then filtered, and the filtrate was precipitated by adding to alarge excess of hexane. The crude monomer was purified by dissolving indichloromethane and precipitating in hexane. Yield 69%.

EXAMPLE 2

[0133] Synthesis of PEG 18.4 kD Tetraacrylate

[0134] A tetrafunctional water soluble PEG (30 g; m.w. 18.5 kD) havingthe following structure was purchased from Polysciences, Inc.:

[0135] where

[0136] F₁=CONH, COO or NHCOO

[0137] X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOH

[0138] F₂=COO, CONH, O or C₆H₄, AND

[0139] R=CH₂ or-alkyl-.

[0140] The PEG was dried by dissolving in benzene and distilling off thewater-benzene azeotrope. PEG 18.5 kD (59 g) was dissolved in 300 mL ofbenzene in a 500 mL flask. To this, 3.6 mL of triethylamine and 2.2 mLof acryloyl chloride were added under nitrogen atmosphere and thereaction mixture was refluxed for 2 hours. It was then cooled andstirred overnight. The triethyl amine hydrochloride was separated byfiltration and the copolymer was recovered from filtrate byprecipitating in a large excess of hexane. The polymer was furtherpurified by dissolving in methylene chloride and reprecipitating inhexane. The polymer was dried at 50° C. under vacuum for 1 day. Yield68%.

EXAMPLE 3

[0141] Coating of Islet-containing Alginate-PLL Microspheres by SurfaceDye Adsorption

[0142] The microcapsule interfacial polymerization method was used toform membrane around alginate-PLL microcapsules containing islets.Alginate-PLL coacervated microspheres, containing one or two humanpancreatic islets each, were suspended in a 1.1% CaCl₂ solution andaspirated free of excess solution to obtain a dense plug ofmicrospheres. A solution of ethyl eosin (0.04% w/v) was prepared in a1.1% CaCl₂ solution. This solution was filter-sterilized by passagethrough a 0.45 μm filter. The plug of microspheres was suspended in 10mL of the eosin solution for 2 min to allow uptake of the dye. Themicrospheres were then washed four times with fresh 1.1% CaCl₂ to removeexcess dye. A solution of PEG 18.5 tetraacrylate (2 mL; 23% w/v)containing 100 μL of a 3.5% w/v solution of triethanolamine in HEPESbuffered saline was added to 0.5 mL of these microspheres. Themicrospheres were exposed to argon ion laser light for 30 seconds withperiodic agitation. The suspension of microspheres was uniformly scannedwith the light during this period. The microspheres were then washedwith calcium solution and the process was repeated in order to furtherstabilize the coating.

[0143] A static glucose stimulation test (SGS) was performed on isletsencapsulated in the microspheres coated with PEG gel. Data for insulinsecretion in response to this challenge appears in Table 1. The isletswere seen to be viable by dithizone staining. The SGS test data confirmthe vitality and functionality of the islets. TABLE 1 SGS initial pulsesubsequent 60 300 60 Glucose Concentration (mg %) Insulin/Islet/hr(μU/mL)* Diffusion Overcoat Method 1.0 10.04 ± 3.56 2.5450.76 MineralOil Overcoat Method 1.0 10.23 ± 3.28 1.0250.78 Free Islet Control 1.03.74 ± 1.4 1.950.17

[0144] PEG diacrylate macromers may be polymerized identically as thePEG tetraacrylate macromer described in this example.

EXAMPLE 4

[0145] Coating Islet-containing Alginate-PLL Microspheres by theMicrocapsule Suspension Polymerization Method

[0146] This method takes advantage of the hydrophilic nature of PEGmonomers. Alginate/PLL microspheres (2 mL), containing one or two humanpancreatic islets each, were mixed with PEG tetraacrylate macromersolution (PEG mol wt 18.5 kD, 23% solution in saline) in a 50 mLtransparent centrifuge tube. Triethanolamine (0.1M) and 0.5 mM ethyleosin were mixed with macromer solution. The excess of macromer solutionwas decanted, 20 mL of mineral oil was added to the tube, and thereaction mixture was vortexed thoroughly for 5 minutes. Silicone oilwill perform equally well in this synthesis but may have poorer adjuvantcharacteristics if there is any carry-over. Any other water-immiscibleliquid may be used as the “oil” phase. Acceptable triethanolamineconcentrations range from about 1 mM to about 100 mM. Acceptable ethyleosin concentrations range from about 0.01 mM to more than 10 mM.

[0147] The beads were slightly red due to the thin coating ofmacromer/dye solution, and they were irradiated for 20-50 sec with anargon ion laser (power 50-500 mW). Bleaching of the (red) ethyl eosincolor suggested completion of the reaction. The beads were thenseparated from mineral oil and washed several times with salinesolution. The entire procedure was carried out under sterile conditions.

[0148] A schematic representation of the microsphere coating process inoil is shown in FIG. 3. Alginate/polylysine capsules are soluble insodium citrate at pH 12. When these coated microspheres came in contactwith sodium citrate at pH 12, the inner alginate/polylysine coacervatedissolves and a PEG polymeric membrane could still be seen (crosslinkedPEG gels are substantially insoluble in all solvents including water andsodium citrate at pH 12). The uncoated control microspheres dissolvedcompletely and rapidly in the same solution.

[0149] A static glucose challenge was performed on the islets as inExample 3. Data again appear in Table 1. The islets were seen to beviable and functional.

EXAMPLE 5

[0150] Encapsulation of Islets of Langerhans

[0151] This example makes use of the direct interfacial polymerization.Islets of Langerhans isolated from a human pancreas were encapsulated inPEG tetraacrylate macromer gels. 500 islets suspended in RPMI 1640medium containing 10% fetal bovine serum were pelleted by centrifugingat 100 g for 3 min. The pellet was resuspended in 1 mL of a 23% w/vsolution of PEO 18.5 kD diacrylate macromer in HEPES buffered saline. Anethyl eosin solution (5 μL) in vinyl pyrrolidone (at a concentration of0.5%) was added to this solution along with 100 μL of a 5 M solution oftriethanolamine in saline. Mineral oil (20 mL) was then added to thetube which was vigorously agitated to form a dispersion of droplets200-500 μm in size. This dispersion was then exposed to an argon ionlaser with a power of 250 mW, emitting at 514 nm, for 30 sec. Themineral oil was then separated by allowing the microspheres to settle,and the resulting microspheres were washed twice with PBS, once withhexane and finally thrice with media.

[0152]FIG. 4 shows islets of Langerhans encapsulated in a PEO gel. Theviability of the islets was verified by an acridine orange and propidiumiodide staining method and also by dithizone staining. In order to testfunctional normalcy, an SGS test was performed on these islets. Theresponse of the encapsulated islets was compared to that of free isletsmaintained in culture for the same time period. All islets weremaintained in culture for a week before the SGS was performed. Theresults are summarized in Table 2. It can be seen that the encapsulatedislets secreted significantly (p<0.05) higher insulin than the freeislets. The PEO gel encapsulation process did not impair function of theislets and in fact helped them maintain their function in culture betterthan if they had not been encapsulated. TABLE 2 Islet Insulin secretion60 300 60 Glucose Concentration (ma %) Insulin/Islet/hr (μU/mL)* Freeislets 1.0  3.74 +/− 1.40 1.9 +/− 0.17 Encapsulated Islets 1.0 20.81 +/−9.36 2.0 +/− 0.76

EXAMPLE 6

[0153] Microencapsulation of Animal Cells

[0154] PEG diacrylates of different molecular weight were synthesized bya reaction of acryloyl chloride with PEG as in Example 1. A 20 to 30%solution of macromer was mixed with a cell suspension and the ethyleosin and triethanolamine initiating system before exposing it to laserlight through a coextrusion air flow apparatus, FIG. 5. Microsphereswere prepared by an air atomization process in which a stream ofmacromer was atomized by an annular stream of air. The air flow rateused was 1,600 cc/min and macromer flow-rate was 0.5 mL/min. Thedroplets were allowed to fall to a petri dish containing mineral oil andwere exposed to laser light for about 0.15 sec each to causepolymerization and make them insoluble in water. Microspheres so formedwere separated from the oil and thoroughly washed with PBS buffer toremove unreacted macromer and residual initiator. The size and shape ofmicrospheres was dependent on extrusion rate (0.05 to 0.1 mL/min) andextruding capillary diameter (18 Ga to 25 Ga). The polymerization timeswere dependent on initiator concentration (ethyl eosin concentration (5μM to 0.5 mM), vinyl pyrrolidone concentration (0.0% to 0.1%),triethanolamine concentration (5 to 100 mM), laser power (10 mw to 1 W),and macromer concentration (>10% w/v).

[0155] A PEG diacrylate macromer of molecular weight 400 Da was used asa 30% solution in PBS, containing 0.1M triethanolamine as a cocatalystand 0.5 mM ethyl eosin as a photoinitiator. Spheres prepared using thismethod are shown in FIG. 6. The polymerizations were carried out atphysiological pH in the presence of air. This is significant sinceradical polymerizations may be affected by the presence of oxygen, andthe acrylate polymerization is still rapid enough to proceedeffectively.

[0156] The process also works at lower temperatures. For cellularencapsulation, a 23% solution of PEO diacrylate was used with initiatingand polymerization conditions as used in the air atomization technique.Cell viability subsequent to encapsulation was checked by trypan blueexclusion assay. Human foreskin fibroblasts (HFF), Chinese hamster ovarycells (CHO-Kl), and a beta cell insuloma line (RiN5F) were found to beviable (more than 95%) after encapsulation. A wide range (>10%) of PEGdiacrylate concentrations may be used equally effectively, as may PEGtetraacrylate macromers.

EXAMPLE 7

[0157] Coating of Animal Cell-containing Alginate-PLL Microspheres andIndividual Cells by Surface Dye Adsorption

[0158] Alginate-PLL coacervated microspheres, containing animal cells,were suspended in a 1.1% CaCl₂ solution and were aspirated free ofexcess solution to obtain a dense plug of microspheres. A solution wasfilter sterilized by passage through a 0.45 pm filter. The plug ofmicrospheres was suspended in 10 mL of eosin solution for 2 min to allowdye uptake. A solution of PEG 18.5 tetraacrylate (2 mL; 23% w/v)containing 100 μL of a 3.5 w/v solution of triethanolamine in HEPESbuffered saline was added to 0.5 mL of these microspheres. Themicrospheres were exposed to an argon ion laser for 30 seconds withperiodic agitation. The suspension of microspheres was uniformly scannedwith the laser during this period. The microspheres were then washedwith calcium solution and the process was repeated once more in order toattain a stable coating.

[0159] In order to verify survival of cells after the overcoat process,cells in suspension without the alginate/PLL microcapsule were exposedto similar polymerization conditions. 1 mL of lymphoblastic leukemiacells (RAJI) (5×10⁵ cells) was centrifuged at 300 g for 3 min. A 0.04%filter sterilized ethyl eosin solution in phosphate buffered saline(PBS) (1 mL) was added and the pellet was resuspended. The cells wereexposed to the dye for 1 min and washed twice with PBS and thenpelleted. Triethanolamine solution (10 μL; 0.1M) was added to the pelletand the tube was vortexed to resuspend the cells. 0.5 mL of PEO 18.5 kDtetraacrylate macromer was then mixed along with this suspension and theresulting mixture was exposed to an argon ion laser (514 nm, 50 mW) for45 sec. The cells were then washed twice with 10 mL saline and once withmedia (RPMI 1640 with 10% FCS and 1% antibiotic, antimycotic). A thinmembrane of PEO gel may be observed forming around each individual cell.

[0160] No significant difference in viability was seen between thecontrol population (93% viable) and the treated cells (95% viable) bytrypan blue exclusion. An assay for cell viability and function wasperformed by adapting the MTT-Formazan assay for the RAJI cells. Thisassay indicates >90% survival. Similar assays were performed with twoother model cell lines. Chinese hamster ovary cells (CHO-Kl) show nosignificant difference (p<0.05) in metabolic function as evaluated bythe MTT-Formazan assay. 3T3 mouse fibroblasts also show no significantreduction (p<0.05) in metabolic activity.

EXAMPLE 8

[0161] Coating Animal Cell Containing Alginate-VLL Microsphers by theOil Suspension Method

[0162] Using the method described in Example 4, RAJI cells contained inalginate-PLL microspheres were coated with a PEG polymeric membrane.Viability of these cells was checked by trypan blue exclusion and theywere found to be more than 95% viable.

EXAMPLE 9

[0163] Coating of Individual Islets of Langerhans by Surface DyeAdsorption

[0164] Using the method described in Example 7, ethyl cosin was adsorbedto the surfaces of islets, exposed to a solution of the PEG macromerwith triethanolamine, and exposed to light from an argon-ion laser toform a thin PEG polymeric membrane on the surface of the islets. Isletviability was demonstrated by lack of staining with propidium iodide.

EXAMPLE 10

[0165] Biocompatibility of PEQ on Microspheres

[0166] In vivo evaluation of the extent of inflammatory response tomicrospheres prepared in Examples 7 and 8 was carried out byimplantation in the peritoneal cavity of mice. Approximately 0.5 mL ofmicrospheres were suspended in 5 mL of sterile HEPES buffered saline. Aportion of this suspension (2.5 mL) was injected into the peritonealcavity of ICR male Swiss white mice. The microspheres were recoveredafter 4 days by conducting a lavage of the peritoneal cavity with 5 mLof 10 U heparin/mL PBS. The extent of cellular growth on themicrospheres was visually inspected under a phase contrast microscope.The number of unattached cells present in the recovered lavage fluid wascounted using a Coulter counter. FIG. 7A shows a photograph ofalginate-poly(L-lysine) microspheres explanted after 4 days, while FIG.7B shows similar spheres which had been coated with PEG gel by the dyediffusion precess before implantation. As expected, bilayeralginate-polylysine capsules not containing an outer alginate layer toprovide an extreme test of the ability of the PEG gel layer to enhancethe biocompatibility of the bilayer membrane, were completely coveredwith cells due to the highly cell adhesive nature of the PLL surface,whereas the PEG coated microspheres were virtually free of adherentcells. Almost complete coverage of alginate-poly(L-lysine) was expectedbecause polylysine has amino groups on the surface, and positivelycharged surface amines can interact with cell surface proteoglycans andsupport cell growth (Reuveny, et al., 1983). The photographs in FIG. 7Bstrongly indicate that the highly charged and cell adhesive surface ofPLL is covered by a stable layer of PEG gel. The integrity of the geldid not appear to be compromised.

[0167] The non-cell-adhesive tendency of these microspheres wasevaluated as a percentage of the total microsphere area which appearscovered with cellular overgrowth. These results are summarized in Table3. TABLE 3 Microsphere Coverage with Cell Overgrowth Composition of PEGgel % Cell coverage 18.5 kD <1 18.5 kD 90%:0.4 kD 10% <1 18.5 kD 50%:0.4kD 50% <1 35k 90%:0.4 kD 10% 5-7 35k 50%:0.4 kD 50% <1 Alginatepoly(L-lysine) 60-80

[0168] An increase in cell count was a result of activation of residentmacrophages which secrete chemical factors such as interleukins andinduce nonresident macrophages to migrate to the implant site. Thefactors also attract fibroblasts responsible for collagen synthesis. Thevariation of cell counts with chemical composition of the overcoat isshown FIGS. 8(A-F). It can be seen from the figure that all PEG coatedspheres have substantially reduced cell counts. This is consistent withthe PEG overcoat generally causing no irritation of the peritonealcavity.

[0169] However, PEG composition does make a difference inbiocompatibility, and increasing molecular weights were associated witha reduction in cell counts. This could be due to the gels made fromhigher molecular weight oligomers having higher potential for stericrepulsion due to the longer chain lengths.

EXAMPLE 11

[0170] Permeability of PEO Gels

[0171] Bovine serum albumin, human IgG, or human fibrinogen (20 mg) wasdissolved in 2 mL of a 23% w/v solution of oligomeric PEO 18.5 kDtetraacrylate in PBS. This solution was laser polymerized to produce agel 2 cm×2 cm×0.5 cm in size. The diffusion of bovine serum albumin,human IgG and human fibrinogen (mol wt 66 kD, 150 kD and 350 kDrespectively) was monitored through the 2 cm×2 cm face of these gelsusing a total protein assay reagent (Biorad). A typical release profilefor a PEO 18.5 kD gel is shown in FIG. 9. This gel allowed a slowtransport of albumin but did not allow IgG and fibrinogen to diffuse.This indicates that these gels are capable of being used asimmunoprotective barriers. This is a vital requirement for a successfulanimal tissue microencapsulation material.

[0172] The release profile was found to be a function of crosslinkdensity and molecular weight of the polyethylene glycol segment of themonomer. FIG. 10 shows the release of BSA through gels made from 23%solutions of PEO diacrylates and tetraacrylates of 0.4 kD and 18.5 kD,respectively. It is evident that the 18.5 kD gel is freely permeable toalbumin while the 0.4 kD gel restricted the diffusion of albumin. Therelease of any substance from these gels will depend on the crosslinkdensity of the network and will also depend on the motility of the PEGsegments in the network. This effect is also dependent upon thefunctionality of the macromer. For example, the permeability of a PEG18.5 kD tetraacrylate gel is less than that of an otherwise similar PEG20 kD diacrylate gel.

[0173] In the case of short PEO chains between crosslinks, the “pore”produced in the network will have relatively rigid boundaries and willbe relatively small and so a macromolecule attempting to diffuse throughthis gel will be predominantly restricted by a sieving effect. If thechain length between crosslinks is long, the chain can fold and movearound with a high motility and, besides the sieving effect, a diffusingmacromolecule will also encounter a free volume exclusion effect.

[0174] Due to these two contrasting effects a straightforward relationbetween molecular weight cutoff for diffusion and the molecular weightof the starting oligomer is not completely definable. Yet, a desiredrelease profile for a particular protein or a drug such as a peptide maybe accomplished by adjusting the crosslink density and length of PEGsegments. Correspondingly, a desired protein permeability profile may bearranged to permit the diffusion of nutrients, oxygen, carbon dioxide,waste products, hormones, growth factors, transport proteins, andreleased cellularly synthesized proteins, while restricting thediffusion of antibodies and complement proteins and also the ingress ofcells, to provide immunoprotectivity to transplanted cells or tissue.The three dimensional crosslinked covalently bonder polymeric network ischemically stable for long-term in vivo applications.

EXAMPLE 12

[0175] Treatment of Silicone Rubber to Enhance Biocompatibility

[0176] Pieces of medical grade silicone rubber (2×2 cm) were soaked for1 h in benzene containing 23% 0.4 kD PEG diacrylate and 0.5%2,2-dimethoxy-2-phenyl acetophenone. The thus swollen rubber wasirradiated for 15 min with a long wave UV lamp (365 nm). Afterirradiation, the sample was rinsed in benzene and dried. The air contactangles of silicone rubber under water were measured before and aftertreatment. The decreased contact angle of 500 after treatment, over theinitial contact angle of 630 for untreated silicone rubber reflects anincreased hydrophilicity due to the presence of the PEG gel on therubber surface.

[0177] This technique demonstrates that macromer polymerization can beused to modify a polymer surface so as to enhance biocompatability. Forinstance, a polyurethane catheter can be treated by this method toobtain an implantable device coated with PEG. The PEG was firmlyanchored to the surface of the polyurethane catheter because themacromer was allowed to penetrate the catheter surface (to a depth of1-2 microns) during the soaking period before photopolymerization. Uponirradiation, an interpenetrating network of PEG and polyurethaneresults. The PEG was thereby inextricably intertwined with thepolyurethane.

EXAMPLE 13

[0178] Treatment of Polyurethane

[0179] INTRACATH (Becton Dickinson) polyurethane intravenous catheters(19 ga) were modified at their outer surfaces with polyethylene glycoldiacrylate (PEG DA) of molecular weight 400 and 10000. The prepolymerwas dissolved in tetrahydrofuran (THF), a solvent for the polyurethane,at 50° C., where polyurethane dissolution is relatively slow. Thefollowing solution was prepared and warmed to 50° C.: PEG DA (MW 400)15% PEG DA (MW 10000) 15% THF 70%

[0180] with 2,2-dimethoxy, 2-phenyl actophenone at 1.6% of the abovesolution.

[0181] 2.5″ length catheter segments were closed at one end by melting a2 mm length by pressing with a hot metal spatula to from a flat tab.This tab was used to fix the catheter in the vessel wall in subsequentanimal experiments. The catheter was held with forceps at the tab endand dipped in the treatment solution for 1-3 sec, pulled out, and theexcess fluid shaken off. The treated catheter was illuminated with anultraviolet light (Black Ray, 360 nm) for 2-3 min, rotating thecatheter. An untreated control was similarly treated in 70% THF with 30%water replacing the PEG in the treatment solution.

[0182] Following this treatment, both the treated and control catheterswere transferred to 100% methylene chloride to extract unreactedmaterials; this extraction was carried out for 36 hr with solventreplacement every 6 hr. These catheters were then dried and transferredto 70% ethanol, and then into water before use.

[0183] A second composition was also investigated: PEG DA (MW 400) 10%PEG DA (MW 10000) 15% Polyethylene oxide (MW 100,000) 5% THF 70%

[0184] with 2,2-dimethoxy, 2-phenyl acetophenone at 1.6% of the abovesolution.

[0185] In this case, the polyethylene oxide of mw 100,000 was not aprepolymer and was immobilized within the PEG DA matrix by entanglement,rather than by chemical attachment.

[0186] Adult New Zealand male rabbits (7-10 lb) were anesthetized withrompun-acepromazien-ketamine. The animal was shaved on the ventrolateraljugular and the vessel was raised. A catheter was inserted into thevessel with the tab outside, and tied in place via the tab with 4.0nylon to the adventitia. The catheter was inserted 1.5 to 2.0″ into thevessel. The skin incision was closed.

[0187] After a period of 3 days, the animals were euthanized by overdoseof pentobarbital intraperitoneally. The vessel was again raised andflushed with phosphate buffered saline (PBS) to superficially rinse awayblood between the catheter and the vessel wall. Two 500 ml bottles, onefilled with PBS and one with formalin in PBS were hung from an i.v. polescaffold, and the hydraulic differential was used to perfusion fix thevessel. The vessels were removed proximal and distal to the ends of thecatheters.

[0188] The treated catheters were completely wettable, and were veryslippery.

[0189] A total of 12 rabbits were catheterized for 72 hr. Six werecontrol, unmodified catheters. These catheters could not be removed fromthe vessel wall without dissection, i.e. they were tightly incorporatedinto the vessel. These catheters upon removal were red, and the vesselwas barely patent. By contrast, the treated catheters were easilyremovable, the vessels were clearly patent, and the catheters were notred. Under the light microscope, a small amount of white thrombus couldbe seen on both formulations of the catheter coating, with somewhatlesser amounts on the formulation containing the polyethylene oxide100,000.

EXAMPLE 14

[0190] Treatment of Ultrafiltration Membranes

[0191] The processes of Examples described above can be applied to thetreatment of macrocapsular surfaces, such as those used forultrafiltration, hemodialysis and non-microencapsulated immunoisolationof animal tissue. The macrocapsule in this case will usually bemicroporous with a molecular weight cutoff below 70,000 Da. It may be inthe form of a hollow fiber, a spiral module, a flat sheet or otherconfiguration. The surface of such a macrocapsule can easily be modifiedusing the PEO gel coating process to produce a non-fouling,non-thrombogenic, and non-cell-adhesive surface. The coating serves toenhance biocompatibility and to offer additional immunoprotection.Materials which can be modified in this manner include polysulfones,cellulosic membranes, polycarbonates, polyamides, polyimides,polybenzimidazoles, nylons, and poly(acrylonitrile-co-vinyl chloride)copolymers and the like.

[0192] Depending on the physical and chemical nature of the surface avariety of methods can be employed to form biocompatible overcoats.Hydrophilic surfaces can simply be coated by-applying a thin layer of a30% w/v polymerizable solution of PEG diacrylate containing appropriateamounts of dye and amine. Hydrophobic surfaces can be first renderedhydrophilic by gas plasma discharge treatment and the resulting surfacecan then be similarly coated, or they may simply be treated with asurfactant before or during treatment with the PEG diacrylate solution.

EXAMPLE 15

[0193] Treatment of Textured Materials and Hydrogels

[0194] The surface of materials having a certain degree of surfacetexture, such as woven dacron, dacron velour, and expandedpoly(tetrafluoroethylene) (ePTFE) membranes, was treated using thecoating method described herein. Textured and macroporous surfaces allowgreater adhesion of the PEG gel to the material surface. This allows thecoating of relatively hydrophobic materials such as PTFE andpoly(ethylene terepthalate) (PET).

[0195] Implantable materials such as enzymatic and ion sensitiveelectrodes, having a hydrogel (such as poly (HEMA), crosslinkedpoly(vinyl alcohol) and poly(vinyl pyrrolidone)) on their surface, arecoated with the more biocompatible PEO gel in a manner similar to thedye adsorption and polymerization technique used for the alginate-PLLmicrospheres.

EXAMPLE 16

[0196] Treatment of Dense Materials

[0197] The surfaces of dense (e.g., nontextured, nongel) materials suchas polymers (including PET, PTFE, polycarbonates, polyamides,polysulfones, polyurethanes, polyethylene, polypropylene, polystyrene),glass, and ceramics can be treated with PEO gel coatings. Hydrophobicsurfaces were initially treated by a gas plasma discharge to render thesurface hydrophilic. This ensures better adhesion of the PEO gel coatingto the surface. Alternatively, coupling agents may be used to increaseadhesion, as readily apparent to those skilled in the art of polymersynthesis.

EXAMPLE 17

[0198] Rate of Polymerization

[0199] To demonstrate rapidity of gelation in laser-initiatedpolymerizations of multifunctional acrylic monomers, the kinetics of atypical reaction were investigated. Trimethylolpropyl triacrylatecontaining 5×10⁻⁴ M ethyl eosin as a photoinitiator in 10 μmoles ofN-vinyl pyrrolidone per mL of macromer mix and 0.1M of triethanolamineas a cocatalyst, was irradiated with a 500 mW argon ion laser (514 nmwavelength, power 3.05×10⁵ W/m², beam diameter 1 mm, average geldiameter produced 1 mm). A plot of the length of the spike of gel formedby penetration of the laser beam into the gel versus laser irradiationtime is shown in FIG. 11A. The spikes formed as a result of laser lightpenetration into the macromer can be seen in FIG. 11B.

[0200] A 23% w/w solution of various macromers in HEPES buffered salinecontaining 3 μL of initiator solution (300 mg/mL of2,2-dimethoxy-2-phenylacetophenone in N-vinyl pyrrolidone) was used. 100μL of the solution was placed on a glass coverslip and irradiated with alow intensity long wave UV (LWUV) lamp (BlakRay, model 3-100 A withflood). The times required for gelation to occur were noted and aregiven in Table 4. These times were typically in the range of 10 seconds.TABLE 4 Gelling Time Gel Time (sec) Polymer Code (mean ± S.D.) 0.4 kD  6.9 ± 0.5 1 kD 21.3 ± 2.4  6 kD 14.2 ± 0.5  10 kD  8.3 ± 0.2 18.5 kD  6.9 ± 0.1 20 kD  9.0 ± 0.4

[0201] Time periods of about 10-100 ms were sufficient to gel a 300 μmdiameter droplet (a typical size of gel used in microencapsulationtechnology). This rapid gelation, if used in conjunction with properchoice of macromers, can lead to entrapment of living cells in a threedimensional covalently bonded polymeric network. The monochromatic laserlight will not be absorbed by the cells unless a proper chromophore ispresent, and is considered to be harmless if wavelength is more thanabout 400 nm. Exposure to long wavelength ultraviolet light (>360 nm) isharmless at practical intensities and durations.

EXAMPLE 18

[0202] PEO Gel Interactions

[0203] Biocompatibility with HFF (human foreskin fibroblasts) cells wasdemonstrated as follows.

[0204] HFF cells were seeded on PEO 18.5 kD tetraacrylate gels at adensity of 18,000 cells/cm² in Dulbecco's modification of Eagle's mediumcontaining 10% fetal calf serum. The gels were then incubated at 37° C.in a 5% CO₂ environment for 4 hr. At the end of this time the gels werewashed with PBS to remove any non-adherent cells and were observed undera phase contrast microscope at a magnification of 200—. FIG. 12A showsthe growth of these cells on a typical PEG gel as compared to glasssurface (FIG. 12B). The number of attached cells/cm² was found to be510±170 on the gel surfaces as compared to 13,200±3,910 for a controlglass surface. The cells on these gels appeared rounded and were not intheir normal spread morphology, strongly indicating that these gels donot encourage cell attachment.

[0205] Biocompatibility on microspheres was demonstrated as follows.FIG. 13 shows a photograph of microspheres explanted from mice as inExample 10; after 4 days very little fibrous overgrowth was seen. Theresistance of PEG chains to protein adsorption and hence cellular growthwas well documented. Table 5 summarizes the extent of cellularovergrowth seen on these microspheres after 4 day intraperitonealimplants for various PEG diacrylate gels. TABLE 5 PEG Diacrylate forGels (mol wt, Daltons) Extent of Cellular Overgrowth 400  5-10% 1,00015-25% 5,000 3-5% 6,000  2-15% 10,000 10-20% 18,500  4-10%

EXAMPLE 19

[0206] Characterization and Mechanical Analysis of PEO Gels

[0207] Solutions of PEO diacrylates (23% w/v; 0.4 kD, 6 kD, 10 kD) andPEG tetraacrylates (18.5 kD) were used. An initiator solution (10 μL)containing 30 mg/mL of 2,2-dimethoxy-2-phenyl acetophenone invinyl-2-pyrrolidone was used per mL of the macromer solution. Thesolution of initiator containing macromer was placed in a 4.0×1.0×0.5 cmmold and exposed to a long wave ultraviolet lamp (365 nm) forapproximately 10 seconds to induce gelation. Samples were allowed toequilibrate in phosphate buffered saline (pH 7.4) for 1 week beforeanalysis 1 performed.

[0208] A series of “dogbone” samples (samples cut from a slab into theshape of a dogbone, with wide regions at both ends and a narrower longregion in the middle) were cut for ultimate tensile strength tests.Thickness of the samples was defined by the thickness of the sample fromwhich they were cut. These thicknesses ranged from approximately 0.5 mmto 1.75 mm. The samples were 20 mm long and 2 mm wide at a narrow “neck”region. The stress strain tests were run in length control at a rate of4% per second. After each test, the cross, sectional area wasdetermined. Table 6 shows the ultimate tensile strength data. It is seenthat the lower molecular weight macromers in general give stronger gelswhich were less extensible than those made using the higher molecularweight macromers. The PEG 18.5 kD tetraacrylate gel is seen to beanomalous in this series, resulting from the multifunctionality of themacromer and the corresponding higher crosslinking density in theresulting gel. This type of strengthening result could be similarlyachieved with macromers obtained having other than four free radicalsensitive groups, such as acrylate groups. TABLE 6 Gel strength TestsPEO Acrylate Precursor Molecular Weight 0.4 kD 6 kD 10 kD 18.5 kD Stress168 +/− 51 98 +/− 15 33 +/− 7  115 +/− 56  (kPa)* %  8 +/− 3 71 +/− 13110 +/− 9  40 +/− 15 Strain* Slope* 22 +/− 5 1.32 +/− 0.31 0.27 +/− 0.042.67 +/− 0.55

[0209] For the creep tests, eight samples approximately 0.2×0.4×2 cmwere loaded while submersed in saline solution. They were tested with aconstant unique predetermined load for one hour and a small recoveryload for ten minutes. Gels made from PEG diacrylates of 1 kD, 6 kD, and10 kD, and PEG tetraacrylates of 18.5 kD PEO molecular weight were usedfor this study. The 10 kD test was terminated due to a limit error (thesample stretched beyond the travel of the loading frame). The 1 kDsample was tested with a load of 10 g and a recovery load of 0.2 g. The6 kD sample was tested at a load of 13 g with a recovery load of 0.5 g.The 18.5 kD sample was tested at a load of 13 g with a recovery load of0.2 g. The choice of loads for these samples produced classical creepcurves with primary and secondary regions. The traces for creep for the1 kD, 6 kD, and 18.5 kD samples appear in FIGS. 14A-C, respectively.

EXAMPLE 20

[0210] Water Content of PEO Gels

[0211] Solutions of various macromers were made as described above. Gelsin the shape of discs were made using a mold. The solutions (400 μL) wasused for each disc. The solutions were irradiated for 2 minutes toensure thorough gelation. The disc shaped gels were removed and driedunder vacuum at 60° C. for 2 days. The discs were weighed (WI) and thenextracted repeatedly with chloroform for 1 day. The discs were driedagain and weighed (W2). The gel fraction was calculated as W2/W1. Thisdata appears in Table 7.

[0212] Determination of Degree of Hydration

[0213] Subsequent to extraction, the discs were allowed to equilibratewith HBS for 6 hours and weighed (W3) after excess water had beencarefully swabbed away. The total water content was calculated as(W3-W2)×100/W3. The data for gel water contents is summarized in thefollowing table. TABLE 7 Polymer Coat % Total Water % Gel Content  0.4kD — 99.8 ± 1.9   1 kD 79.8 ± 2.1 94.5 ± 2.0   6 kD 95.2 ± 2.5 69.4 ±0.6   10 kD 91.4 ± 1.6 96.9 ± 1.5 18.5 kD 91.4 ± 0.9 80.3 ± 0.9   20 kD94.4 ± 0.6 85.0 ± 0.4

EXAMPLE 21

[0214] Mechanical Stability of PEO Gels after Implantation

[0215] PEG diacrylate (10 kD) and PEG tetraacrylate (18.5 kD) were castin dogbone shapes as described in Example 19. PEG-dacrylate ortetraacrylate (23% w/w) in sterile HEPES buffered saline (HBS) (0.9%NaCl, 10-HEPES, pH 7.4) containing 900 ppm of2,2-dimethoxy-2-phenoxyacetophenone as initiator, was poured into analuminum mold and irradiated with a LWUV lamp (Black ray) for 1 min. Theinitial weights of these samples were found after oven-drying these gelsto constant weight. The samples were soxhlet-extracted with methylenechloride for 36 hours in order to leach out any unreacted prepolymerfrom the gel matrix (sol-leaching) prior to testing. The process ofextraction was continued until the dried gels gave constant weight.

[0216] ICR Swiss male white mice, 6-8 weeks old (Sprague-Dawley), wereanesthetized by an intraperitoneal injection of sodium pentobarbital.The abdominal region of the mouse was shaved and prepared with betadine.A ventral midline incision 10-15 mm long was made. The polymer sample,fully hydrated in sterile PBS (Phosphate buffered saline) or HEPESbuffered saline (for calcification studies), was inserted through theincision and placed over the mesentery, away from the wound site. Theperitoneal wall was closed with a lock stitched running suture (4.0silk, Ethicon). The skin was closed with stainless steel skin staples,and a topical antibiotic (Furacin) was applied over the incision site.Three animals were used for each time point. One dogbone sample wasimplanted per mouse and explanted at the end of 1 week, 3 weeks, 6weeks, and 8 weeks. Explanted gels were rinsed in HBS twice and thentreated with 0.3 mg/mL pronase (Calbiochem) to remove any adherent cellsand tissue. The samples were then oven-dried to a constant weight,extracted, and reswelled as mentioned before.

[0217] Tensile stress strain test was conducted on both control(unimplanted) and explanted dogbones in a small horizontal Instron-likedevice. The device is an aluminum platform consisting of two clampsmounted flat on a wooden board between two parallel aluminum guide. Thetop clamp was stationary while the bottom clamp was movable. Both thefrictional surfaces of the moving clamp and the platform were coatedwith aluminum backed Teflon (Cole-Parmer) to minimize frictionalresistance. The moving clamp was fastened to a device capable ofapplying a gradually increasing load. The whole set up was placedhorizontally under a dissecting microscope (Reichert) and the sampleelongation was monitored using a video camera. The image from the camerawas acquired by an image processor (Argus-10, Hamamatsu) and sent to amonitor. After breakage, a cross section of the break surface was cutand the area measured. The load at break was divided by this crosssection to find the maximum tensile stress. Table 8 lists the stress atfracture of PEG tetraacrylate (18.5 kD) hydrogels explanted at varioustime intervals. No significant change in tensile strength was evidentwith time. Thus, the gels appear mechanically stable to biodegradationin vivo within the maximum time frame of implant in mice. TABLE 8 TIMESTRESS (KPa) STRAIN AV. IMPLANTED (mean ± error*) (mean ± error*) 1 WK52.8 ± 16.7 0.32 ± 0.19 3 WK 36.7 ± 10.6 0.37 ± 0.17 6 WK 73.3 ± 34.90.42 ± 0.26 8 WK 34.1‡ 0.30‡ CONTROL 44.9 ± 5.3  0.22 ± 0.22

EXAMPLE 22

[0218] Monitoring of Calcification of PEO Gels

[0219] Disc shaped PEG-tetraacrylate hydrogels (m.w. 18.5 kD) wereimplanted intraperitoneally in mice as mentioned above for a period of 1week, 3 weeks, 6 weeks, or 8 weeks. Explanted gels were rinsed in HBStwice and treated with Pronase (Calbiochem) to remove cells and celldebris. The samples were then equilibrated in HBS to let free Ca⁺⁺diffuse out from the gel matrix. The gels were then oven-dried (Blue-M)to a constant weight and transferred to Aluminum oxide crucibles (COORS,high temperature resistant). They were incinerated in a furnace at 700°C. for at least 16 hours. Crucibles were checked for total incineration,if any residual remnants or debris was seen they were additionallyincinerated for 12 hours. Subsequently, the crucibles were filled with 2mL of 0.5 M HCl to dissolve Ca⁺⁺ salt and other minerals in the sample.This solution was filtered and analyzed with atomic absorptionspectroscopy (AA) for calcium content.

[0220] Calcification data on PEG-tetraacrylate (mol. wt. 18.5 kD) gelimplants is given in Table 9. No significant increase in calcificationwas observed up to an 8 week period of implantation in mice. TABLE 9TIME CALCIFICATION (mean ± error*) (Days) (mg Calcium/g of Dry gel wt.) 7 2.33 ± 0.20 21  0.88 ± 0.009 42 1.08 ± 0.30 56 1.17 ± 0.26

EXAMPLE 23

[0221] Encapsulation of Neurotransmitter-releasing Cells

[0222] Paralysis agitans, more commonly called Parkinson's disease, ischaracterized by a lack of the neurotransmitter dopamine within thestriatum of the brain. Dopamine secreting cells such as cells from theventral mesencephalon, from neuroblastoid cell lines or from the adrenalmedulla can be encapsulated in a manner similar to that of other cellsmentioned in prior Examples. Cells (including genetically engineeredcells) secreting a precursor for a neurotransmitter, an agonist, aderivative or a mimic of a particular neurotransmitter or analogs canalso be encapsulated.

EXAMPLE 24

[0223] Encapsulation of Hemoglobin for Synthetic Erythrocytes

[0224] Hemoglobin in its free form can be encapsulated in PEG gels andretained by selection of a PEG chain length and cross-link density whichprevents diffusion. The diffusion of hemoglobin from the gels may befurther impeded by the use of polyhemoglobin, which is a cross-linkedform of hemoglobin. The polyhemoglobin molecule is too large to diffusefrom the PEG gel. Suitable encapsulation of either native or crosslinkedhemoglobin may be used to manufacture synthetic erythrocytes. Theentrapment of hemoglobin in small spheres (<5 μm) of these highlybiocompatible materials would lead to enhanced circulation timesrelative to crosslinked hemoglobin or liposome encapsulated hemoglobin.

[0225] Hemoglobin in PBS is mixed with the prepolymer in the followingformulation: Hemoglobin at the desired amount PEG DA (MW 10000) 35% PEGDA (MW 1000)  5% PBS 60%

[0226] with 2,2-dimethoxy, 2-phenyl acetophenone at 1.6% of the abovesolution.

[0227] This solution is placed in mineral oil at a ratio of 1 parthemoglobin/prepolymer solution to 5 parts mineral oil and is rapidlyagitated with a motorized mixer to form an emulsion. This emulsion isilluminated with a long-wavelength ultraviolet light (360 nm) for 5 minto crosslink the PEG prepolymer to form a gel. The mw of the prepolymermay be selected to resist the diffusion of the hemoglobin from the gel,with smaller PEG DA molecular weights giving less diffusion. PEG DA ofMW 10000, further crosslinked with PEG DA 1000, should possess theappropriate permselectivity to restrict hemoglobin diffusion, and itshould possess the appropriate biocompatibility to circulate within thebloodstream.

EXAMPLE 25

[0228] Entrapment of Enzymes for Correction of Metabolic Disorders andChemotherapy

[0229] Congenital deficiency of the enzyme catalase causes acatalasemia.Immobilization of catalase in PEG gel networks could provide a method ofenzyme replacement to treat this disease. Entrapment of glucosidase cansimilarly be useful in treating Gaucher's disease. Microspherical PEGgels entrapping urease can be used in extracorporeal blood to converturea into ammonia. Enzymes such as asparaginase can degrade amino acidsneeded by tumor cells. Immunogenicity of these enzymes prevents directuse for chemotherapy. Entrapment of such enzymes in immunoprotective PEGgels, however, can support successful chemotherapy. A suitableformulation can be developed for either slow release or no release ofthe enzyme.

[0230] Catalase in PBS is mixed with the prepolymer in the followingformulation: Catalase at the desired amount PEG DA (MW 10000) 35% PEG DA(MW 1000)  5% PBS 60%

[0231] with 2,2-dimethoxy, 2-phenyl acetophenone at 1.6% of the abovesolution.

[0232] This solution is placed in mineral oil at a ratio of 1 partcatalase/prepolymer solution to 5 parts mineral oil and is rapidlyagitated with a motorized mixer to form an emulsion. This emulsion inilluminated with a long-wavelength ultraviolet light (360 nm) for 5 minto crosslink the PEG prepolymer to form a gel. The mw of the prepolymermay be selected to resist the diffusion of the catalase from the gel,with smaller PEG DA molecular weights giving less diffusion.

[0233] PEG DA of MW 10,000, further crosslinked with PEG DA 1000, shouldpossess the appropriate permselectivity to restrict catalase diffusion,and it should possess the appropriate permselectivity to permit thediffusion of hydrogen peroxide into the gel-entrapped catalase to allowthe enzymatic removal of the hydrogen peroxide from the bloodstream.Furthermore, it should possess the appropriate biocompatibility tocirculate within the bloodstream.

[0234] In this way, the gel is used for the controlled containment of abioactive agent within the body. The active agent (enzyme) is large andis retained within the gel, and the agent upon which it acts (substrate)is small and can diffuse into the enzyme rich compartment. However, theactive agent is prohibited from leaving the body or targeted bodycompartment because it cannot diffuse out of the gel compartment.

EXAMPLE 26

[0235] Cellular Microencapsulation for Evaluation of Anti-humanImmunodeficiency Virus Drugs in vivo

[0236] HIV infected or uninfected human T-lymphoblastoid cells can beencapsulated into PEG gels as described for other cells above. Thesemicrocapsules can be implanted in a nonhuman animal and then treatedwith test drugs such as AZT or DDI. After treatment, the microcapsulescan be harvested and the encapsulated cells screened for viability andfunctional normalcy using a fluorescein diacetate/ethidium bromidelive/dead assay. Survival of infected cells indicates successful actionof the drug. Lack of biocompatibility is a documented problem in thisapproach to drug evaluations, but the highly biocompatible gelsdescribed herein solve this problem.

EXAMPLE 27

[0237] Use of PEG Gels as Adhesive to Rejoin Severed Nerve

[0238] A formulation of PEG tetraacrylate (10%, 18.5 kD), was used asadhesive for stabilizing the sutureless apposition of the ends oftransected sciatic nerves in the rat. Rats were under pentobarbitalanesthesia during sterile surgical procedures. The sciatic nerve wasexposed through a lateral approach by deflecting the heads of the bicepsfemoralis at the mid-thigh level. The sciatic nerve was mobilized forapproximately 1 cm and transected with iridectomy scissors approximately3 mm proximal to the tibeal-peroneal bifurcation. The gap between theends of the severed nerves was 2-3 mm. The wound was irrigated withsaline and lightly swabbed to remove excess saline. Sterile,unpolymerized PEG tetraacrylate solution was applied to the wound. Usingdelicate forceps to hold the adventitia or perineurium, the nerve endswere brought into apposition, the macromer solution containing2,2-dimethoxy-2-phenylacetophenone as a photoinitiator and the wound wasexposed to long wavelength UV-light (365 nm) for about 10 sec topolymerize the adhesive. The forceps were gently pulled away. Care wastaken to prevent the macromer solution from flowing between the twonerve stumps. Alternatively, the nerve stump junction was shielded fromillumination, e.g. with a metal foil, to prevent gelation of themacromer solution between the stumps; the remaining macromer solutionwas then simply washed away.

[0239] In an alternative approach, both ends of the transected nervewere held together with one pair of forceps. Forcep tips were coatedlightly with petrolatum to prevent reaction with the adhesive. Thepolymerized adhesive serves to encapsulate the wound and adhere thenerve to the underlying muscle. The anastomosis of the nerve endsresists gentle mobilization of the joint, demonstrating a moderatedegree of stabilization. The muscle and skin were closed with sutures.Reexamination after one month shows that severed nerves can remainreconnected, despite unrestrained activity of the animals.

EXAMPLE 28

[0240] Surgical Adhesive

[0241] The unpolymerized macromer mixture was an aqueous solution, suchas that of PEO 400 kD diacrylate or PEO 18.5 kD tetraacrylate. When thissolution contacts tissue which has a moist layer of mucous or fluidcovering it, it intermixes with the moisture on the tissue. The mucouslayer on tissue comprises water soluble polysaccharides which intimatelycontact cellular surfaces. These, in turn, are rich in glycoproteins andproteoglycans. Thus, physical intermixing, hydrogen bonding, and forcesof surface interlocking due to penetration into crevices are some of theforces responsible for the adhesion of the PEO gel to a tissue surfacesubsequent to crosslinking.

[0242] PEO diacrylate solutions can therefore be used as tissueadhesives as in the previous example. Specific applications for suchadhesives may include blood vessel anastomosis, soft tissuereconnection, drainable burn dressings, and retinal reattachment.However, if the PEO gel is polymerized away from tissue, it thenpresents a very non-adhesive surface to cells and tissue in general, dueto the low interfacial energy intrinsic to the material.

[0243] Abdominal muscle flaps from female New Zealand white rabbits wereexcised and cut into strips 1 cm×5 cm. The flaps were approximately 0.5to 0.8 cm thick. A lap joint (1 cm×1 cm) was made using two such flaps.Two different PEO di- and (tetra-) acrylate macromer compositions, 0.4kD (di-) and 18.5 kD (tetra-), were evaluated. The 0.4 kD compositionwas a viscous liquid and was used without further dilution. The 18.5 kDcomposition was used as a 23% w/w solution in HBS. 125 μL of ethyl eosinsolution in n-vinyl pyrrolidone (20 mg/mL) along with 50 μL oftriethanolamine was added to each mL of the adhesive solution. 100 μL ofadhesive solution was applied to each of the overlapping flaps. The lapjoint was then irradiated by scanning with a 2 W argon ion laser for 30seconds from each side. The strength of the resulting joints wasevaluated by measuring the force required to shear the lap joint. Oneend of the lap joint was clamped and an increasing load was applied tothe other end, while holding the joint horizontally until it failed.Four joints were tested for each composition. The 0.4 kD joints had astrength of 12.0±6.9 KPa (mean±S.D.), while the 18.5 kD joints had astrength of 2.7±0.5 KPa. It is significant to note that it was possibleto achieve photopolymerization and reasonable joint strength despite the6-8 mm thickness of tissue. A spectrophotometric estimate using 514 nmlight showed less than 1% transmission through such muscle tissue.

EXAMPLE 29

[0244] Modification of PVA Polymer

[0245] Water soluble polymers other than PEG can also be modified usinga similar chemical approach to that in prior examples. Thus, poly(vinylalcohol) (PVA), which is water soluble, can be easily crosslinked togive a gel wherein the crosslinking occurs under conditions mild enoughto permit gelation in contact with or in the vicinity of biologicalmaterials such as tissues, mammalian cells, proteins, orpolysaccharides. The method adopted herein leads to PVA crosslinkingunder very mild conditions as compared to the standard techniques offormaldehyde crosslinking.

[0246] Polyvinyl alcohol (2 g; m.w. 100,000-110,000 D) was dissolved in20 mL of hot DMSO. The solution was cooled to room temperature, and 0.2mL of triethylamine and 0.2 mL of acryloyl chloride was added withvigorous stirring under an argon atmosphere. The reaction mixture washeated to 70° C. for 2 hr and cooled. The polymer was precipitated inacetone, redissolved in hot water, and precipitated again in acetone.Finally, it was dried under vacuum for 12 hr at 60° C. A solution ofthis polymer in PBS (5-10% w/v) was mixed with the UV photoinitiator andpolymerized using long wavelength UV light to make microspheres200-1,000 microns in size. These microspheres were stable to autoclavingin water, which indicates that the gel was covalently cross-linked. Thegel was extremely elastic. This macromer, PVA multiacrylate, may be usedto increase the crosslinking density in PEG diacrylate gels, withcorresponding changes in mechanical and permeability properties. Thisapproach could be pursued with any number of water-soluble polymerswhich are chemically modified with photopolymerizable groups; forexample, with water-soluble polymers chosen from polyvinylpyrrolidone,polyethyloxazoline, polyethyleneoxide-polypropyleneoxide copolymers,polysaccharides such as dextran, alginate, hyaluronic acid, chondroitinsulfate, heparin, heparin sulfate, heparan sulfate, guar gum, gel Iangum, xanthan gum, carrageenan gum, etc., and proteins such as albumin,collagen, gelatin, and the like.

EXAMPLE 30

[0247] Use of Alternative Photopolymerizable Moieties

[0248] Many photopolymerizable groups may be used to enable gelation.Several possibilities are shown below, based upon a PEG central chain:

[0249] where

[0250] F₁=CONH, COO or NHCOO

[0251] X=H, CH₃, C₂H₅, Cl, Br, OH or CH₂COOH

[0252] F₂=COO, CONH, O or C₆H₄,

[0253] R=CH₂ or-alkyl-,

[0254] n≧5, and

[0255] m≧3.

[0256] To illustrate a typical alternative synthesis, a synthesis forPEG 1 kD urethane methacrylate is described as follows, where F₁═NHCOO,F₂═COO, R═CH₃ and X═CH₃:

[0257] In a 250 mL round bottom flask, 10 g of PEG 1 kD was dissolved in150 mL benzene. 2-isocyanatoethylmethacrylate (3.38 g) and 20 pL ofdibutyltindilaurate were slowly introduced into the flask. The reactionwas refluxed for 6 hours, cooled, and poured into 1000 mL hexane. Theprecipitate was filtered and dried under vacuum at 60° C. for 24 hours.

EXAMPLE 31

[0258] Use of Alternative Photoinitiator/Photosensitizer Systems

[0259] It is possible to initiate photopolymerization with a widevariety of dyes as initiators and a number of electron donors aseffective cocatalysts. Table 10 illustrates photopolymerizationinitiated by several other dyes which have chromophores absorbing atwidely different wavelengths. All gelations were carried out using a 23%w/w solution of 18.5 kD PEG tetraacrylate in HEPES buffered saline.These initiating systems compare favorably with conventional thermalinitiating systems, as can also be seen from Table 10. TABLE 10Polymerization Initiation TEMPER- APPROXIMATE LIGHT ATURE GEL TIME,INITIATOR SOURCE* ° C. (SEC) Eosin Y, 0.00015M, S1 with UV 25 10Triethanolamine 0.65M filter Eosin Y, 000015M; S4 25 0.1 Triethanolamine0.65M Methylene Blue, S3 25 120 0.00024M; p- toluenesulfonic acid,0.0048M 2,2-dimethoxy-2-phenyl S2 25 8 acetophenone 900 ppm PotassiumPersulfate — 75 180 0.0168M Potassium Persulfate — 25 120 0.0168M;tetramethyl ethylene-diamine 0.039M Tetramethyl ethylene- S1 with UV 25300 diamine 0.039M; filter Riboflavin 0.00047 M

[0260] CODE SOURCE S1 Mercury lamp, LEITZ WETZLER Type 307-148.002, 100WS2 Black Ray longwave UV lamp, model B-100A W/FLOOD S3 MELLES GRIOTHe-Ne laser, 10 mW output, 1=632 nm S4 American laser corporation, argonion laser, model 909BP-15-01001; 1=485 and 514 nm

[0261] Numerous other dyes can be used for photopolymerization. Thesedyes include but are not limited to Erythrosin, phloxime, rose bengal,thionine, camphorquinone, ethyl eosin, eosin, methylene blue, andriboflavin. Possible cocatalysts that can be used include but are notlimited to: N-methyl diethanolamine, N,N-dimethyl benzylamine,triethanolamine, triethylamine, dibenzyl amine, N-benzyl ethanolamine,N-isopropyl benzylamine.

EXAMPLE 32

[0262] Formation of Alginate-PLL-Alginate Microcapsules withPhotopolymerizable Polycations

[0263] Alginate-polylysine-alginate microcapsules were made byadsorbing, or coacervating, a polycation, such as polylysine (PLL), upona gelled microsphere of alginate. The resulting membrane was heldtogether by charge-charge interactions and thus has limited stability.To increase this stability, the polycation can be madephotopolymerizable by the addition of carbon-carbon double bonds, forexample. This can be used to increase the stability of the membrane byitself, or to react, for example, with photopolymerizable PEG to enhancebiocompatibility.

[0264] To illustrate the synthesis of such a photopolymerizablepolycation, 1 g of polyallylamine hydrochloride was weighed in 100 mLglass beaker and dissolved in 10 mL distilled water (DW). The pH of thepolymer solution was adjusted to 7 using 0.2M sodium hydroxide solution.The polymer was then separated by precipitating in a large excess ofacetone. It was then redissolved in 10 mL DW and the solution wastransferred to 50 mL round bottom flask. Glycidyl methacrylate (0.2 mL)was slowly introduced into the reaction flask and the reaction mixturewas stirred for 48 hours at room temperature. The solution was pouredinto 200 mL acetone and the precipitate was separated by filtration anddried in vacuum.

[0265] In addition to use in encapsulating cells in materials such asalginate, such photopolymerizable polycations may be useful as a primeror coupling agent to increase polymer adhesion to cells, cellaggregates, tissues, and synthetic materials, by virtue of adsorption ofthe photopolymerizable polymer bonding to the PEG photopolymerizablegel.

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We claim:
 1. A method for encapsulation of biological materialcomprising the steps of a) mixing the biological material in an aqueousmacromer solution comprising macromer and photoinitiator; b) formingsmall globular geometric shapes of the mix in (a); and c) polymerizingthe macromer by exposing the geometric shapes to light radiation.
 2. Themethod of claim 1 wherein the macromer is a water soluble, ethylenicallyunsaturated, polymer susceptible to polymerization into an waterinsoluble polymer through interaction of at least two carbon-carbondouble bonds.
 3. The method of claim 2 wherein the macromer is selectedfrom the group consisting of ethylenically unsaturated derivatives ofpoly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG), poly(vinylalcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline)(PEOX), poly(amino acids), polysaccharides, and proteins.
 4. The methodof claim 3 wherein the PEG is PEG multiacrylate.
 5. The method of claim4 wherein the PEG is PEG tetraacrylate which has a molecular weightaround 18,500 D.
 6. The method of claim 3 wherein the polysaccharidesare selected from the group consisting of alginate, hyaluronic acid,chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate,heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, watersoluble cellulose derivatives and carrageenan.
 7. The method of claim 3wherein the proteins are selected from the group consisting of gelatin,collagen and albumin.
 8. The method of claim 1 wherein the macromer isof the formula

where F₁=CONH, COO or NHCOO X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOHF₂=COO, CONH, O or C₆H₄, R=CH₂ or-alkyl-, n≧5, and m≧2.
 9. The method ofclaim 1 wherein the photoinitiator is any dye which absorbs light havingfrequency between 320 nm and 900 nm, can form free radicals, is at leastpartially water soluble, and is non-toxic to the biological material atthe concentration used for polymerization.
 10. The method of claim 9wherein the photoinitiator is selected from the group consisting of2,2-dimethoxy,2-phenyl-acetophenone and 2-methoxy,2-phenylacetophenone.11. The method of claim 1 wherein the macromer solution furthercomprises a cocatalyst and the photoinitiator is selected from the groupconsisting of ethyl eosin, eosin Y, fluorescein,2,2-dimethoxy,2-phenylacetophenone, 2-methoxy,2-phenylacetophenone,camphorquinone, rose bengal, methylene blue, erythrosin, phloxime,thionine, riboflavin and methylene green.
 12. The method of claim 11wherein the cocatalyst is a nitrogen based compound capable ofstimulating a free radical reaction.
 13. The method of claim 11 whereinthe cocatalyst is a a nitrogen atom-containing electron-rich molecule.14. The method of claim 11 wherein the cocatalyst is a primary,secondary, tertiary or quaternary amine.
 15. The method of claim 14wherein the cocatalyst is selected from the group consisting oftriethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine,N,N-dimethyl benzylamine, dibenzyl amine, N-benzyl ethanolamine,N-isopropyl benzylamine, tetramethyl ethylenediamine, potassiumpersulfate, tetramethyl ethylenediamine, lysine, ornithine, histidineand arginine
 16. The method of claim 1 wherein the radiation has awavelength between 320 nm and 900 nm.
 17. The method of claim 16 whereinthe radiation has a wavelength between 350 nm and 700 nm.
 18. The methodof claim 1 wherein the biological material is selected from mammaliantissue, mammalian cells, sub-cellular organelles and sub-cellularnon-organelle components.
 19. The method of claim 18 wherein the cellsare primary cells or established cell lines.
 20. The method of claim 18wherein the biological material is selected from pancreatic islet cells,human foreskin fibroblasts, Chinese hamster ovary cells, beta cellinsulomas, lymphoblastic leukemia cells, mouse 3T3 fibroblasts, dopaminesecreting ventral mesencephalon cells, neuroblastoid cells, adrenalmedulla cells, and T-cells.
 21. The method of claim 1 wherein thebiological material is selected from proteins, polysaccharides,oligonucleotides, enzymes, enzyme systems, bacteria, microbes, vitamins,cofactors, blood clotting factors, drugs, immunogens, hormones, andretroviruses.
 22. The method of claim 21 wherein the protein ishemoglobin.
 23. The method of claim 21 wherein the enzyme is adenosinedeaminase.
 24. The method of claim 21 wherein the drugs are selectedfrom TPA, streptokinase and heparin.
 25. The method of claim 1 whereinthe geometric shapes are formed by coextrusion of the aqueous macromersolution mixed with the biological material with a non-toxic,non-immunogenic, non-miscible substance capable of maintaining dropletformation.
 26. The method of claim 25 wherein the non-miscible substanceis oil
 27. The method of claim 26 wherein the oil is mineral oil. 28.The method of claim 1 wherein the geometric shapes are formed bycoextrusion of the aqueous macromer solution mixed with the biologicalmaterial in air.
 29. The method of claim 1 wherein the geometric shapesare formed by agitation of the aqueous macromer solution mixed with thebiological material with a non-toxic, non-immunogenic, non-misciblesubstance.
 30. The method of claim 29 wherein the non-miscible substanceis oil.
 31. The method of claim 1 wherein the biological material isfirst encapsulated in a microcapsule.
 32. The method of claim 31 whereinthe microcapsule is comprised of ionically coagulatable or thermallycoagulatable polymers which are non-toxic to the encapsulated material.33. The method of claim 32 wherein the microcapsule is comprised ofalginate.
 34. The method of claim 32 wherein the microcapsule iscomprised of chitosan.
 35. The method of claim 32 wherein themicrocapsule is comprised of agarose.
 36. The method of claim 32 whereinthe microcapsule is comprised of gelatin.
 37. The method of claim 1wherein the macromer solution further comprises an accelerator toaccelerate the rate of polymerization.
 38. The method of claim 37wherein the accelerator is a small molecule containing an allyl, vinylor acrylate group.
 39. The method of claim 38 wherein the accelerator isselected from the group consisting of N-vinyl pyrolidinone, 2-vinylpyridine, 1-vinyl imidazole, 9-vinyl carbazole, acrylic acid and2-allyl,2-methyl,1-3-cyclopentane dione.
 40. The method of claim 39wherein the accelerator is N-vinyl pyrolidinone.
 41. A method forencapsulation of biological material comprising the steps of a) coatingthe biological material with photoinitiator; b) suspending the coatedmaterial in a macromer solution comprised of macromer; and c)irradiating the suspension with light.
 42. The method of claim 41wherein the macromer is a water soluble, ethylenically unsaturated,polymer susceptible to polymerization into an water insoluble polymerthrough interaction of at least two carbon-carbon double bonds.
 43. Themethod of claim 42 wherein the macromer is selected from the groupconsisting of ethylenically unsaturated derivatives of poly(ethyleneoxide) (PEO), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline) (PEOX), poly(aminoacids), polysaccharides, and proteins.
 44. The method of claim 43wherein the PEG is PEG multiacrylate.
 45. The method of claim 44 whereinthe PEG is PEG tetraacrylate which has a molecular weight around 18,500D.
 46. The method of claim 43 wherein the polysaccharides are selectedfrom the group consisting of alginate, hyaluronic acid, chondroitinsulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparansulfate, chitosan, gellan gum, xanthan gum, guar gum, water solublecellulose derivatives and carrageenan.
 47. The method of claim 43wherein the proteins are selected from the group consisting of gelatin,collagen and albumin.
 48. The method of claim 41 wherein the macromer isof the formula

where F₁=CONH, COO or NHCOO X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOHF₂=COO, CONH, O or C₆H₄, R=CH₂ or-alkyl-, n≧5, and m≧2.
 49. The methodof claim 41 wherein the photoinitiator is any dye which absorbs lighthaving frequency between 320 nm and 900 nm, can form free radicals, isat least partially water soluble, and is non-toxic to the biologicalmaterial at the concentration used for polymerization.
 50. The method ofclaim 49 wherein the photoinitiator is selected from the groupconsisting of 2,2-dimethoxy,2-phenyl-acetophenone and2-methoxy,2-phenylacetophenone.
 51. The method of claim 41 wherein themacromer solution further comprises a cocatalyst and the photoinitiatoris selected from the group consisting of ethyl eosin, eosin Y,fluorescein, 2,2-dimethoxy,2-phenylacetophenone,2-methoxy,2-phenylacetophenone, camphorquinone, rose bengal, methyleneblue, erythrosin, phloxime, thionine, riboflavin and methylene green.52. The method of claim 51 wherein the cocatalyst is a nitrogen basedcompound capable of stimulating a free radical reaction.
 53. The methodof claim 51 wherein the cocatalyst is a a nitrogen atom-containingelectron-rich molecule.
 54. The method of claim 51 wherein thecocatalyst is a primary, secondary, tertiary or quaternary amine. 55.The method of claim 54 wherein the cocatalyst is selected from the groupconsisting of triethanolamine, triethylamine, ethanolamine, N-methyldiethanolamine, N,N-dimethyl benzylamine, dibenzyl amine, N-benzylethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine,potassium persulfate, tetramethyl ethylenediamine, lysine, ornithine,histidine and arginine.
 56. The method of claim 41 wherein the radiationhas a wavelength between 320 nm and 900 nm.
 57. The method of claim 56wherein the radiation has a wavelength between 350 nm and 700 nm. 58.The method of claim 41 wherein the biological material is selected frommammalian tissue, mammalian cells, sub-cellular organelles andsub-cellular non-organelle components.
 59. The method of claim 58wherein the cells are primary cells or established cell lines.
 60. Themethod of claim 58 wherein the biological material is selected frompancreatic islet cells, human foreskin fibroblasts, Chinese hamsterovary cells, beta cell insulomas, lymphoblastic leukemia cells, mouse3T3 fibroblasts, dopamine secreting ventral mesencephalon cells,neuroblastoid cells, adrenal medulla cells, and T-cells.
 61. The methodof claim 41 wherein the biological material is selected from proteins,polysaccharides, oligonucleotides, enzymes, enzyme systems, bacteria,microbes, vitamins, cofactors, blood clotting factors, drugs,immunogens, hormones, and retroviruses.
 62. The method of claim 61wherein the protein is hemoglobin.
 63. The method of claim 61 whereinthe enzyme is adenosine deaminase.
 64. The method of claim 61 whereinthe drugs are selected from TPA, streptokinase and heparin.
 65. Themethod of claim 41 wherein the geometric shapes are formed bycoextrusion of the aqueous macromer solution mixed with the biologicalmaterial with a non-toxic, non-immunogenic, non-miscible substancecapable of maintaining droplet formation.
 66. The method of claim 65wherein the non-miscible substance is oil.
 67. The method of claim 66wherein the oil is mineral oil.
 68. The method of claim 41 wherein thegeometric shapes are formed by coextrusion of the aqueous macromersolution mixed with the biological material in air.
 69. The method ofclaim 41 wherein the geometric shapes are formed by agitation of theaqueous macromer solution mixed with the biological material with anon-toxic, non-immunogenic, non-miscible substance.
 70. The method Ofclaim 69 wherein the non-miscible substance is oil.
 71. The method ofclaim 41 wherein the biological material is first encapsulated in amicrocapsule.
 72. The method of claim 71 wherein the microcapsule iscomprised of ionically coagulatable or thermally coagulatable polymerswhich are non-toxic to the encapsulated material.
 73. The method ofclaim 72 wherein the microcapsule is comprised of alginate.
 74. Themethod of claim 72 wherein the microcapsule is comprised of chitosan.75. The method of claim 72 wherein the microcapsule is comprised ofagarose.
 76. The method of claim 72 wherein the microcapsule iscomprised of gelatin.
 77. The method of claim 41 wherein the macromersolution further comprises an accelerator to accelerate the rate ofpolymerization.
 78. The method of claim 77 wherein the accelerator is asmall molecule containing an allyl, vinyl or acrylate group.
 79. Themethod of claim 78 wherein the accelerator is selected from the groupconsisting of N-vinyl pyrolidinone, 2-vinyl pyridine, 1-vinyl imidazole,9-vinyl carbazole, acrylic acid and 2-allyl,2-methyl,1-3-cyclopentanedione.
 80. The method of claim 79 wherein the accelerator is N-vinylpyrolidinone.
 81. A method of applying a biocompatible surface to abiomedical device having a polymeric surface which can be at leastpartly swelled comprising the steps of: (a) swelling the polymericsurface in a solvent; (b) applying a macromer solution, comprised ofmacromer, to the surface; (c) irradiating the macromer solution toinitiate polymerization; and (d) deswelling the polymeric surface byremoving it from the solvent.
 82. The method of claim 81 wherein themacromer is a water soluble, ethylenically unsaturated, polymersusceptible to polymerization into an water insoluble polymer throughinteraction of at least two carbon-carbon double bonds.
 83. The methodof claim 82 wherein the macromer is selected from the group consistingof ethylenically unsaturated derivatives of poly(ethylene oxide) (PEO),poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline) (PEOX), poly(aminoacids), polysaccharides, and proteins.
 84. The method of claim 83wherein the PEG is PEG multiacrylate.
 85. The method of claim 84 whereinthe PEG is PEG tetraacrylate which has a molecular weight around 18,500D.
 86. The method of claim 83 wherein the polysaccharides are selectedfrom the group consisting of alginate, hyaluronic acid, chondroitinsulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparansulfate, chitosan, gellan gum, xanthan gum, guar gum, water solublecellulose derivatives and carrageenan.
 87. The method of claim 83wherein the proteins are selected from the group consisting of gelatin,collagen and albumin.
 88. The method of claim 81 wherein the macromer isof the formula

where F₁=CONH, COO or NHCOO X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOHF₂=COO, CONH, O or C₆H₄, R=CH₂ or-alkyl-, n≧5, and m≧2.
 89. The methodof claim 81 wherein polymerization is initiated by gamma ray or electronbeam radiation.
 90. The method of claim 81 wherein the macromer solutionfurther comprises a photoinitiator, and polymerization is initiated bylight having wavelength of 320-900 nm.
 91. The method of claim 90wherein the photoinitiator is any dye which absorbs light havingfrequency between 320 nm and 900 nm, can form free radicals, is at leastpartially water soluble, and is non-toxic to the biological material atthe concentration used for polymerization.
 92. The method of claim 91wherein the photoinitiator is selected from the group consisting of2,2-dimethoxy,2-phenyl-acetophenone and 2-methoxy,2-phenylacetophenone.93. The method of claim 81 wherein the macromer solution furthercomprises a cocatalyst and the photoinitiator is selected from the groupconsisting of ethyl eosin, eosin Y, fluorescein,2,2-dimethoxy,2-phenylacetophenone, 2-methoxy,2-phenylacetophenone,camphorquinone, rose bengal, methylene blue, erythrosin, phloxime,thionine, riboflavin and methylene green.
 94. The method of claim 93wherein the cocatalyst is a nitrogen based compound capable ofstimulating a free radical reaction.
 95. The method of claim 93 whereinthe cocatalyst is a a nitrogen atom-containing electron-rich molecule.96. The method of claim 93 wherein the cocatalyst is a primary,secondary, tertiary or quaternary amine.
 97. The method of claim 96wherein the cocatalyst is selected from the group consisting oftriethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine,N,N-dimethyl benzylamine, dibenzyl amine, N-benzyl ethanolamine,N-isopropyl benzylamine, tetramethyl ethylenediamine, potassiumpersulfate, tetramethyl ethylenediamine, lysine, ornithine, histidineand arginine.
 98. The method of claim 81 wherein the radiation has awavelength between 320 nm and 900 nm.
 99. The method of claim 98 whereinthe radiation has a wavelength between 350 nm and 700 nm.
 100. Themethod of claim 81 wherein the macromer solution further comprises anaccelerator to accelerate the rate of polymerization.
 101. The method ofclaim 100 wherein the accelerator is a small molecule containing anallyl, vinyl or acrylate group.
 102. The method of claim 101 wherein theaccelerator is selected from the group consisting of N-vinylpyrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazole,acrylic acid and 2-allyl,2-methyl,1-3-cyclopentane dione.
 103. Themethod of claim 102 wherein the accelerator is N-vinyl pyrolidinone.104. The method of claim 81 wherein the biomedical device is a catheter.105. The method of claim 81 wherein the biomedical device is a containerenclosing mammalian tissue or cells.
 106. The method of claim 81 whereinthe biomedical device is tubing.
 107. The method of claim 81 wherein thebiomedical device is an ultrafiltration membrane.
 108. A method forjoining together two biological surfaces comprised of forming a waterinsoluble polymer between and adhering to each of the two surfacescomprising the steps of: (a) juxtaposing the two surfaces to be joined;(b) applying to the joint a macromer solution comprised of macromer andphotoinitiator; and (c) polymerizing the macromer by exposing themacromer solution to light radiation.
 109. The method of claim 108wherein the macromer is a water soluble, ethylenically unsaturated,polymer susceptible to polymerization into an water insoluble polymerthrough interaction of at least two carbon-carbon double bonds.
 110. Themethod of claim 109 wherein the macromer is selected from the groupconsisting of ethylenically unsaturated derivatives of poly(ethyleneoxide) (PEO), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline) (PEOX), poly(aminoacids), polysaccharides, and proteins.
 111. The method of claim 110wherein the PEG is PEG multiacrylate.
 112. The method of claim 111wherein the PEG is PEG tetraacrylate which has a molecular weight around18,500 D.
 113. The method of claim 110 wherein the polysaccharides areselected from the group consisting of alginate, hyaluronic acid,chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate,heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, watersoluble cellulose derivatives and carrageenan.
 114. The method of claim110 wherein the proteins are selected from the group consisting ofgelatin, collagen and albumin.
 115. The method of claim 108 wherein themacromer is of the formula

where F₁=CONH, COO or NHCOO X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOHF₂=COO, CONH, O or C₆H₄, R=CH₂ or-alkyl-, n≧5, and m≧2.
 116. The methodof claim 108 wherein the photoinitiator is any dye which absorbs lighthaving frequency between 320 nm and 900 nm, can form free radicals, isat least partially water soluble, and is non-toxic to the biologicalmaterial at the concentration used for polymerization.
 117. The methodof claim 116 wherein the photoinitiator is selected from the groupconsisting of 2,2-dimethoxy,2-phenyl-acetophenone and2-methoxy,2-phenylacetophenone.
 118. The method of claim 108 wherein themacromer solution further comprises a cocatalyst and the photoinitiatoris selected from the group consisting of ethyl eosin, eosin Y,fluorescein, 2,2-dimethoxy,2-phenylacetophenone,2-methoxy,2-phenylacetophenone, camphorquinone, rose bengal, methyleneblue, erythrosin, phloxime, thionine, riboflavin and methylene green.119. The method of claim 118 wherein the cocatalyst is a nitrogen basedcompound capable of stimulating a free radical reaction.
 120. The methodof claim 119 wherein the cocatalyst is a a nitrogen atom-containingelectron-rich molecule.
 121. The method of claim 118 wherein thecocatalyst is a primary, secondary, tertiary or quaternary amine. 122.The method of claim 121 wherein the cocatalyst is selected from thegroup consisting of triethanolamine, triethylamine, ethanolamine,N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amine,N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethylethylenediamine, potassium persulfate, tetramethyl ethylenediamine,lysine, ornithine, histidine and arginine.
 123. The method of claim 108wherein the radiation has a wavelength between 320 nm and 900 nm. 124.The method of claim 123 wherein the radiation has a wavelength between350 nm and 700 nm.
 125. The method of claim 108 wherein the macromersolution further comprises an accelerator to accelerate the rate ofpolymerization.
 126. The method of claim 125 wherein the accelerator isa small molecule containing an allyl, vinyl or acrylate group.
 127. Themethod of claim 126 wherein the accelerator is selected from the groupconsisting of N-vinyl pyrolidinone, 2-vinyl pyridine, 1-vinyl imidazole,9-vinyl carbazole, acrylic acid and 2-allyl,2-methyl,1-3-cyclopentanedione.
 128. The method of claim 127 wherein the accelerator is N-vinylpyrolidinone.